International Immunology Advance Access originally published online on December 21, 2007
International Immunology 2008 20(2):165-175; doi:10.1093/intimm/dxm133
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Selective silencing of disease-associated B-lymphocytes by chimeric molecules targeting their Fc
IIb receptor
1 Department of Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
2 Central Laboratory of Immunology, National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
3 Department of Pathology, Sofia Medical School, Sofia, Bulgaria
4 Department of Gene Regulation, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Correspondence to: T. Vassilev, Stefan Angelov Institute of Microbiology, Academy G. Bonchev Street, Block 26, 1113 Sofia, Bulgaria. E-mail: vassilev{at}microbio.bas.bg
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionFundingReferences |
|---|
The presently used approaches to silence autoreactive disease-associated B cells act indiscriminately and more specific therapies are obviously needed. In the present study, we analyze the ability of a chimeric antibody to suppress selectively pathological autoreactive B-lymphocytes in lupus-prone mice by cross-linking their surface Ig receptors with the inhibitory IgG Fc
RIIb receptors. The chimera was constructed by coupling an immunodominant mouse Histone 1 peptide to a rat monoclonal anti-mouse CD32 (FcRIIb) antibody. The administration of these chimeric molecules to MRL/lpr mice with initial and with full-blown disease resulted in the reduction of the levels of IgG anti-Histone 1 antibodies, of the albuminuria levels, of the size of lymphoid organs and in prevention of the development of skin lesions. The observed effect was limited to lupus-associated B cells only, as the treatment did not decrease the IgG antibody response to an administered foreign antigen. This study demonstrates the possibility to silence selectively autoreactive B cells and to delay the progression of an autoimmune disease using chimeric antibody molecules.
Keywords: auto-antibodies, FcRIIb, Histone 1, lupus
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionFundingReferences |
|---|
Autoreactive B cells have a dual role in the pathogenesis of antibody-mediated autoimmune diseases. First, they are precursors of the plasma cells that produce pathological auto-antibodies and second, they act as efficient antigen-presenting cells that help brake tolerance to self-antigens in autoreactive T-lymphocytes (1–3). The selective suppression and/or elimination of the autoreactive disease-associated B cells are logical goals in the efforts to control autoimmunity.
Depletion of B cells by the infusion of an anti-CD20 mAb (Rituximab) has had a beneficial effect in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis and other autoimmune diseases that do not respond to conventional treatment (4–7). Immunotherapy with unconjugated anti-CD19 mAb also results in a rapid elimination of pre-B, immature B cells and mature B-lymphocytes in animals (8). Both B-cell depletion treatments eliminate for long periods most B cells regardless of their antigen specificity. This is a serious disadvantage that underlines the necessity to develop specific approaches for the selective silencing of disease-associated B-lymphocytes.
It is known that B-1 lymphocytes give rise to most DNA IgG-producing plasma cells in NZB/W mice. The selective elimination of these cells since an early age was shown to prevent the development of glomerulonephritis and to increase dramatically the lifespan of the animals (9). It has been also demonstrated that B-cell superantigens [(SpA) from Staphylococcus aureus] can reduce B-1 cell numbers in vivo. Viau and Zouali (10) have found that weekly intra-peritoneal injections of SpA delayed the increase of serum anti-DNA IgG levels and reduced proteinuria in young female (NZB x NZW)F1 mice with initial disease. However, these immunomodulatory approaches are not effective in lupus-prone MRL/lpr mice because B-1 cells are not involved in the pathogenesis of the disease.
Toll Like Receptor 9, a receptor for hypomethylated CpG DNA motifs, is expressed by human and mouse B cells. It has been implicated in the breakdown of immunologic tolerance to DNA in SLE. The injection of a synthetic G-rich DNA (used as an antagonist of CpG-rich DNA) to MRL (lpr/lpr) mice with glomerulonephritis suppressed specifically the CpG–DNA-induced proliferation of B cells. The treatment with G-rich DNA also reduced IgG2a anti-dsDNA antibody levels as well as the glomerular immune complex deposits (11).
Reiners et al. (12) reported the construction of a chimeric toxin, killing B cells specific for proteinase 3—the target antigen in Wegener’s disease. Zocher et al. (13) have designed a bi-specific fusion protein composed of the extracellular Ig-like domain of human myelin oligodendrocyte glycoprotein (MOG) and of the CH2 and CH3 domains of the human IgG1 heavy chain (MOG-Fc). A selective in vivo and in vitro elimination of MOG-specific B-lymphocytes was observed. Treatment of MRL/lpr mice with pre-formed DNA–anti-DNA immune complexes has resulted in suppression of the production of disease-associated auto-antibodies and reduction of the severity of lupus nephritis (14). Immune complexes, however, could hardly be used for therapy because of numerous standardization, stability and shelf-life problems.
SLE is a systemic autoimmune disease characterized by the generation of IgG auto-antibodies specific to nuclear antigens—double-stranded (ds) DNA (15), histones (16), nucleosomes (17), etc. The primary auto-antigen that drives the autoimmune response in SLE is not known. DNA in its pure form is not immunogenic. Nucleosomes from apoptotic cells have been shown to contain the self-antigens that induce disease-associated antibodies in (SWR x NZB)F1 (SNF1) lupus-prone mice (18). Kaliyaperumal et al. (19) have eluted a number of histone peptides from MHC class II molecules of an antigen-presenting cell line fed with crude chromatin from lupus-prone SNF1 mice. One of these peptides (H1'22–42) is part of the Histone 1 molecule. H1'22–42 has been shown to be a potent stimulator of autoimmune Th and accelerated lupus nephritis when administrated to SNF1 mice. Antibodies, specific to the H1'22–42 peptide, at later stages of the disease bound also to other nuclear antigens.
The activity of an individual B lymphocyte depends on the balance between the positive and negative signals from its surface receptors. A number of inhibitory receptors—CD22, CD32 (FcRIIb) and CD72 (20)—on B cells prevent undesirable responses. Deficiency of the inhibitory FcR (FcRIIb) on B cells results in imbalanced immune responses and in the development of autoimmune pathology (21, 22). FcRIIb plays an important role in maintaining immune tolerance in the periphery (21, 23). IgG-containing immune complexes provide a negative feedback signal to the antigen-specific B cells by cross-linking their inhibitory FcRIIb receptors with the IgG B cell receptors (BCRs). This suppressing signal blocks the proliferation and differentiation of the targeted B-lymphocytes (24).
We hypothesize that it should be possible to suppress the activity of pre-selected B cells in vivo by administering a chimeric molecule able to cross-link FcRIIb with autoreactive BCRs on their surface. In the present study, we show that a chimeric molecule, constructed by coupling copies of the natural H1'22–42 peptide to an anti-mouse FcRIIb-binding mAb suppresses selectively Histone 1-specific B-lymphocytes in lupus-prone MRL/lpr mice.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionFundingReferences |
|---|
Mice
MRL/MpJ-Faslpr/J mice were purchased from Harlan Farm, Blackthorn, UK, and were kept under specific pathogen-free conditions. The experimental protocols used were approved by the Animal Care Commission at the Institute of Microbiology in accordance with the International Regulations.
Antibodies
The rat 2.4G2 hybridoma producing a monoclonal IgG2b antibody specific for mouse CD32 (FcRII, ATCC HB-197) and the rat hybridoma I/9 producing a monoclonal anti-idiotype [anti-B10-anti-(T,G-A-L)] IgG2b antibody [(25) kindly provided by Gloria Laszlo, Immunology Research Group, Hungarian Academy of Sciences, Budapest, Hungary] were adapted to grow in the serum-free CHO medium (Gibco, Gaithersburg, MD, USA). The antibodies from the supernatant were isolated by 50% ammonium sulfate precipitation and subsequent dialysis. Their purity was determined by 10% SDS–PAGE under non-reducing conditions using a silver staining kit (PlusOne, Pharmacia Biotech AB, Uppsala, Sweden) and by blotting using an anti-rat kappa-chain alkaline phosphatase conjugate (this and all other commercial mAbs used in this study, if not stated otherwise, were purchased from PharMingen BD, San Diego, CA, USA). Care was taken to diminish all possible sources of endotoxin during the purification and subsequent conjugation steps. A commercial FITC conjugate of the 2.4G2 antibody was also used.
The mouse 10F10 hybridoma producing an anti-dsDNA IgG antibody (kindly provided by Bor-Luen Chiang, College of Medicine, National Taiwan University, Taipei, Taiwan) was cultured and the mAb was obtained as described above.
Conjugation of the chimeric antibody molecules
Two synthetic peptides—the H1'22–42 peptide (STDHPKYSDMIVAAIQAEKNR) and an irrelevant peptide, containing the same amino acids, but in a shuffled order (PDAASRMAETYHKDSVQNHK)—were used in the study. The synthesis of Ac-STDHPKYSDMIVAAIQAEKNR-NH-(CH2)6-NH2 and of Ac-PDAASRMAETYHKDSVQNHK-NH-(CH2)6-NH2 was carried out using Fmoc-based manual solid-phase peptide synthesis protocols on 2-Cl-Trt resin. The peptides were purified (to 
95% purity) by reversed phase sample displacement chromatography (26) on a series of 3 x 50 mm Nucleodur 100-5 C18 columns (Macherey-Nagel, Dueren, Germany). The H1'22–42 peptide was coupled to the 2.4G2 antibody (2.4G2-H1'22–42 chimera) and to the irrelevant antibody I/9 (control chimera 2). The irrelevant peptide was coupled to the 2.4G2 antibody (control chimera 1).
The coupling of the antibodies to the peptides was carried out using the classical 1-ethyl-3(3'-dimethylaminopropyl) carbodiimide·HCl (Fluka AG, Buchs, Switzerland) cross-linking technique (27, 28). During the synthesis of the peptides, a spacer [H2N(CH2)6H2N] was added to their C-end that enables the peptides to reach their conformation properly. To avoid cross-linking of the Ig molecules, their concentration was kept low. The antibody (in concentration 0.1 mg ml–1 in sterile 0.1 M sodium phosphate buffer, pH 6.0) was mixed with a 20-fold molar excess of the peptide [dissolved in 10% (v/v) N,N-dimethylformamide (Sigma–Aldrich, Taufkirchen, Germany) in the same buffer to 0.02 mg ml–1 final concentration). The reaction was started by the addition of carbodiimide at 60-fold molar excess over the antibody. The reaction mixture was stirred overnight at 4°C, dialyzed against PBS and concentrated by ultrafiltration.
Flow cytometry
Spleen cell suspensions from female MRL/lpr mice were prepared by grinding through sterile cell mesh; erythrocytes were lysed with a hypotonic ammonium chloride solution. The binding of the 2.4G2-H1'22–42 chimera to mouse spleen cells was compared with that of the 2.4G2 antibody itself. Cells were washed with PBS (containing 2.5% FCS and 0.05% sodium azide) and incubated with the 2.4G2 antibody or with the 2.4G2-H1'22–42 chimera for 30 min at 4°C, followed by two washes. Next, the cells were incubated with goat anti-rat kappa light-chain biotin-conjugated antibody (PharMingen BD), followed by incubation with FITC-conjugated avidin (Calbiochem, Darmstadt, Germany).
In a separate experiment, the same cells were incubated with biotinylated anti-CD19 antibody and subsequently with streptavidin–PE (Sigma–Aldrich, St.Louis, MO, USA). On the next step, the cells were incubated with several dilutions of the 2.4G2-H1'22–42 chimera (10, 2.5, 0.5 and 0.1 µg 10–6 cells) for 30 min at 4°C followed by washing and incubation with FITC-conjugated 2.4G2 antibody (PharMingen BD). Each incubation step was performed for 30 min at 4°C. Finally, the cells were washed twice and kept at 4°C for not >1 h until analyzed with a FACSCanto flow cytometer (BD Biosciences). Ten thousand cells were collected for each sample.
The cell population, targeted by the 2.4G2-H1'22–42 chimera, was characterized by flow cytometry. For B-cell binding, the following approach was used: splenocytes from a sick MRL/lpr mouse were incubated with the chimeric construct and with biotinylated anti-mouse CD19 antibody, washed and incubated further with FITC-conjugated anti-rat IgG antibody and streptavidin–PE. To study T-cell binding, splenocytes were treated with the chimera and a hamster anti-mouse CD3 antibody after washing with anti-hamster IgG–FITC (from Autogen Bioclear, Calve, UK) and biotinylated anti-rat IgG. After a new washing step, staining with streptavidin–PE followed.
The total number of B- and T-lymphocytes was also measured. Splenocytes from a disease-free (7 weeks old), an untreated sick and a chimera-treated sick 16-week-old MRL/lpr mice were prepared as described above and stained with anti-mouse CD3–FITC, CD19–FITC as well as PE-conjugates of anti-mouse CD24, CD27 and CD38 (all from PharMingen BD).
Assay for apoptosis
Expression of phosphatidylserine was determined in gated CD19+ and CD3+ spleen cells from a sick MRL/lpr mice and pre-cultured for 3 days in the presence of increasing concentrations of the constructed chimeric molecules. At that point, the splenocytes were washed, stained with PE/Cy5-conjugated anti-CD19 or CD3 antibodies and with Annexin V–FITC and propidium iodide using the Annexin V–FITC apoptosis detection kit (BD Biosciences PharMingen).
Cross-blotting
The H1'22–42 peptide was coupled to ovalbumin (as described above). Two 3-month-old chinchilla rabbits were immunized subcutaneously (s.c.) with 100 µg of this conjugate emulsified in CFA and boosted three times at intervals of 4 weeks with the same antigen in IFA. The immune serum with the highest level of anti-H1 antibodies was used further. To prove the presence of the H1'22–42 peptide in the 2.4G2-H1'22–42 chimera conjugate and in the control chimera 2, we used a cross-blotting technique that has been described in detail elsewhere (29). Briefly, a nitrocellulose membrane (0.45 µm, from Sartorius, Goettingen, Germany) was incubated with anti-rabbit IgG antibody (AbD Serotec, Raleigh, NC, USA) for 60 min at room temperature (RT). After extensive washing, the membrane was blocked with Tris-buffered saline (TBS)/Tween 20 (0.2%) overnight at 4°C and inserted in a mini-blotter cassette (Miniblotter 28 SL, Immunetics, Camridge, MA, USA). Serial rabbit serum dilutions were added to the individual slots for 60 min at RT. The membrane was taken out, washed with TBS/Tween 20 (0.05%) and returned to the mini-blotter cassette in a direction perpendicular to that of the imprints of the slots containing the rabbit serum during the first incubation step. It was further incubated with serial dilutions of the 2.4G2-H1'22–42 chimera, the control chimera 1, the control chimera 2, the unconjugated 2.4G2 antibody or Histone 1 for 60 min at RT. After washing, the membrane was incubated for 1 h with mouse anti-rat IgG (H+L) (Biomeda Corporation, Foster City, CA, USA), followed by a HRP-labeled goat anti-mouse IgG (H+L) (Novagen, San Diego, CA, USA).
Treatment schedule
Groups of 7-week-old female MRL/lpr mice with initial disease and groups of 18-week-old ones with severe disease (10 mice per group) were injected intravenously (i.v.), twice weekly for 8 weeks with 20 µg of the H1'22–42 peptide chimera, with the same amount of the control chimeras or with PBS alone. Every 14 days, the animals were bled and their sera kept frozen at –20°C.
Measurement of proteinuria levels
Proteinuria was measured every 2 weeks using Combi-screen strips (Analyticon Biotechnologies, Lichenfels, Germany) and graded semi-quantitatively (0, none; 1, 30–100; 2, 100–300; 3, 300–500 and 4, >500 mg dl–1).
ELISA for anti-Histone 1 antibodies
ELISA for anti-Histone 1 antibodies was performed as previously described (16) with modifications. Briefly, mouse Histone 1 [obtained as shown in (30)] was dissolved in PBS, pH 7.2 at 10 µg ml–1 and coated onto polystyrene microtiter plates [96-well Maxisorp immunoplates (Nunc, Roskilde, Denmark)] at a volume of 100 µl per well and incubated overnight at 4°C. The plates were washed and blocked with 0.1% gelatine in PBS–Tween 20 (0.05%) for 2 h at RT, followed by three washes. Diluted serum samples (diluted 1:100 in PBS) were then added to duplicate wells and incubated for 1 h at RT. After five washes, the plates were incubated with peroxidase-conjugated anti-mouse IgG ( chain specific) antibody (PharMingen BD) for 1 h at RT. After the final washing step, the reaction was visualized by the addition of an 2,2'-azino-bis(3-ethyl benz-thiazoline-6-sulfonic acid) solution (Sigma, Munich, Germany) containing H2O2 and read at 405 nm. For determination of anti-Histone 1 antibodies belonging to individual IgG subclasses, alkaline phosphatase-conjugated antibodies to mouse IgG subclasses (IgG1, IgG2a, IgG2b and IgG3 from PharMingen BD) were used. Experimental optical density (OD) values from separate experiments were normalized to a single MRL/lpr-positive control serum used in every assay.
ELISA for anti-DNA antibodies and for other lupus antigens
It was performed as previously described (24). For determination of anti-dsDNA antibody level belonging to individual IgG subclasses, alkaline phosphatase-conjugated antibodies to mouse IgG subclasses (IgG1, IgG2a, IgG2b and IgG3 from PharMingen BD) were used. Experimental OD values from separate experiments were normalized to a supernatant from 10F10 hybridoma as a positive control used in every assay. IgG anti-SSB/La, anti-SSA/Ro and anti-Sm IgG antibodies in sera of immunized animals were detected using commercial ELISA kits (from Alpha Diagnostic International, San Antonio, TX, USA) according to the manufacturer’s instructions.
ELISPOT assays
Spleen cells were obtained from sick MRL/lpr mice 10 days after the second s.c. immunization with 2.5 µg alum-adsorbed diphtheria toxoid. The splenocytes were cultured for 5 days at 2 x 106 cells ml–1 in RPMI 1640 (Gibco) containing 10% FCS, 4 mM L-glutamine, 50 µM 2-mercaptoethanol and antibiotics in the presence of the 2.4G2-H1'22–42 chimera, control chimeras, 10 µg ml–1 LPS (from Escherichia coli; Sigma, L-2630—used as a positive control) or medium alone. Nitrocellulose membranes (0.45 µm, from Sartorius) were coated with 10 µg ml–1 mouse Histone 1, 10 µg ml–1 calf thymus dsDNA or 30 µg ml–1 diphtheria toxoid (from BulBio, Sofia, Bulgaria) for 30 min at RT. The cells from each well were transferred to a well of the manifold apparatus (Bio-Dot, Bio-Rad) on the pre-coated membranes and were incubated for five additional hours in a humidified 5% CO2 atmosphere at 37°C. After washing, the membranes were incubated with Fc-specific anti-mouse IgG conjugated with alkaline phosphatase (Sigma, Germany) for 2 h at RT and developed using the nitroblue tetrazolium/bromo-chloro-indolyl-phosphate chromogenic substrate (Sigma, Germany). The spots were counted blindly under a microscope by independent observers.
Renal histopathology
Kidneys from treated animals were formalin fixed, embedded in a paraffin block, stained with hematoxylin–eosin and observed by light microscopy.
Statistical analysis
Differences between sample means were analyzed using Student's t-test and were considered statistically significant at P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionFundingReferences |
|---|
Characterization of the chimeric molecule conjugates
The functional activity of the two building elements of the designed artificial chimeric molecules was studied. There are many free carboxyl groups on the surface of the antibody molecule but only few of them are available for interaction with the reactive H2N group of the hexamethylene spacer bound to the N-end of the H1'22–42 peptide. Theoretically, these peptides could also bind to groups within the antigen-binding sites and as a result the interaction of the parent antibody with the mouse Fc
RIIb would be affected. We excluded this possibility by comparing the ability of the native 2.4G2 antibody and the 2.4G2-H1'22–42 chimera to recognize the targeted receptor only on gated mouse B-lymphocytes (data not shown). The results showed that the binding sites of the antibody component of the chimera were not modified and retained their antigen-binding activity after the conjugation process. These results were confirmed in a separate experiment in which it was shown that the 2.4G2-H1'22–42 chimera inhibited in a dose-dependent manner the binding of a commercial FITC-labeled 2.4G2 antibody to the spleen B cells (Fig. 1A).
|
Pure heavy and light chains from native 2.4G2 antibody and from the 2.4G2-based chimera were obtained by high-performance liquid chromatography after reducing of the intact Ig molecule. Mass spectral analyses of the 2.4G2-based chimera showed that 14 to 16 peptides were bound per single IgG molecule (Mihaylova, Nikolina et al., submitted for publication).
The recognition of the H1'22–42 peptide by the pooled serum from rabbits, repeatedly immunized with the same peptide (see Methods) in a cross-blot assay (Fig. 1B), confirmed that the H1'22–42 peptides on the surface of the chimeric antibodies retained their B-cell epitope. The latter is available for interaction with Histone 1-specific B-lymphocyte receptors. The ability of the antibody and of the peptide antigen of the 2.4G2-H1'22–42 chimera to bind both—the FcRIIb and the surface Histone 1-specific Ig receptors—on autoreactive B-lymphocytes was preserved. Therefore, the artificial molecule was expected to cross-link the same receptors on the surface of the targeted disease-associated B cells in lupus mice. To characterize the cell population to which the chimeric conjugates bound, spleen cells from a sick MRL/lpr mice were incubated with the chimera and its binding to gated CD19+ and CD3+ cells was studied by FACS analysis. The chimeric conjugate did not bound to T cells but it did bind to a population of B cells in the suspension (Fig. 1C).
Treatment with the 2.4G2-H1'22–42 chimera reduced disease-associated auto-antibody levels
We used two groups of lupus-prone female MRL/lpr mice: 7- and 18-week old animals. The younger mice were still disease-free at that age but the appearance of the first signs of lupus was imminent. The older group had already high levels of IgG anti-Histone 1 and anti-dsDNA antibodies at the start of the experiment. The period of treatment was limited to 8 weeks because at that point anti-rat Ig antibodies appeared in all chimera-injected animals (data not shown).
We found that the administration of the chimeric antibody to 7-week-old mice markedly decreased the levels of anti-Histone 1 IgG antibodies (Fig. 2A, left panel) during the treatment period and prevented the rise of the anti-DNA IgG antibody levels during the first 2 weeks (Fig. 2B, left panel). Treatment started at the age of 18 weeks resulted in maintaining a flat level of IgG anti-Histone 1 antibodies during the following 8 weeks (Fig. 2A, right panel). No effect on the levels of anti-DNA IgG antibodies was observed in this group (Fig. 2B, right panel). In order to exclude the possibility that DNA-containing immune complexes from the tested lupus mice sera could interfere with the ELISA tests for anti-Histone 1 IgG antibodies, we repeated these tests in the presence of increasing concentrations of pre-formed DNA immune complexes. The results from a series of ELISA tests showed that the DNA immune complexes from the animals’ sera did not interfere with the interactions of anti-Histone 1 antibodies from the same sera with the immobilized Histone 1 (data not shown).
|
The levels of anti-Histone 1 and of anti-DNA IgG antibody subclasses in the sera of all groups were also studied. A reduction in the levels of IgG1 and IgG2a anti-Histone 1 antibodies in the sera of young mice treated with the chimeric antibody was observed. An effect of the chimera on the levels of the anti-DNA IgG antibodies was detected only for IgG2a in the sera of the young animals and for IgG3 in the sera of the older ones (data not shown). No differences regarding the levels of IgG antibodies to the SSA, SSB and Sm antigens were established between the groups (data not shown).
The effect of 2.4G2-H1'22–42 chimera was limited to anti-Histone 1- and anti-dsDNA-specific B cells only
Sixteen-week-old MRL/lpr mice with lupus were immunized twice with alum-adsorbed diphtheria toxoid. Ten days after the boosting, they had large numbers of toxoid-specific B-lymphocytes. Their spleen cells were cultured for 5 days and the numbers of anti-Histone 1, anti-DNA and anti-diphtheria toxoid IgG antibody-producing cells were determined by the ELISPOT technique. The addition of bacterial LPS did not further increase the numbers of antibody producers, showing that most B cells with the studied specificities were already activated. The 2.4G2-H1'22–42 chimera caused a dose-dependent decrease of the numbers of anti-Histone 1 (Fig. 3A) and anti-dsDNA IgG antibody-producing cells (Fig. 3B). None of the constructed chimeras added to the medium during the period of cultivation influenced the numbers of diphtheria toxoid-specific IgG antibody-producing cells (Fig. 3C).
|
The 2.4G2-H1'22–42 chimera decreases the size of lymphoid organs and prevents skin lesions in MRL/lpr mice
Groups of 7-week-old female MRL/lpr mice (10 animals per group) were treated twice weekly for 8 weeks with 20 µg of the 2.4G2-H1'22–42 chimera, control chimeras or PBS alone. After the end of the treatment, all control mice had enlarged spleens and lymph nodes (Fig. 4A, right, B, C, top group, and D), whereas the size of these lymphoid organs in the 2.4G2-H1'22–42 chimera-treated animals was smaller (Fig. 4A, left, B, C, bottom group, and D).
|
MRL/lpr mice develop spontaneously cutaneous lesions. No lesions were detected in any of the 2.4G2-H1'22–42 chimera-treated animals (Fig. 4C, right panel) while at 5 months of age, all control mice had developed them on the posterior of their necks (Fig. 4C, left panel and Table 1).
|
It is known that the enlargement of the lymphoid organs in MRL/lpr mice is primarily due to the accumulation of large numbers of T cells. To study the effect of the treatment on the fate of lymphoid cells, we cultured splenocytes for 3 days in the presence of increasing concentrations of the chimeric molecules and measured Annexin V staining. High spontaneous apoptosis was seen in gated B cells, cultured in medium only that was further increased after exposure to the 2.4G2-H1'22–42 chimera (Fig. 5A). The same was true for cultured T cells (data not shown).
|
In separate experiment, the numbers of B and T cells belonging to the different subsets were determined in the spleens from disease-free, untreated sick and chimera-treated sick mice. The dramatic increase of T-cell populations observed in the sick PBS-treated animal (2.71 x 108 per spleen) was prevented in the chimera-treated one (0.4 x 108 per spleen, ). This could well explain the differences between the mean total numbers of the splenocytes in both groups (4.1 x 108 and 1.03 x 108) and close to normal sizes of the spleens of the chimera-treated MRL/lpr mice.
Treatment with 2.4G2-H1'22–42 antibody chimera attenuates renal disease in MRL/lpr mice
The administration of the 2.4G2-H1'22–42 chimera decreased significantly the proteinuria levels through the first weeks of the treatment (Fig. 6A). Kidney sections from control mice (Fig. 6B, top right, bottom left and right) showed severe perivascular inflammatory cell infiltrates, necrotizing and sclerosing lesions and massive mesangial proliferation in most glomeruli. Kidneys from chimera-treated mice had a preserved histological structure in spite of the presence of an interstitial infiltrate (Fig. 6B, top left). Although the administration of the chimera resulted in the suppression of disease-associated IgG antibody levels and of renal and cutaneous involvement, the survival of the animals was not affected (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionFundingReferences |
|---|
There is still no effective therapy targeting selectively disease-associated autoreactive B cells in autoimmune patients. We show in the present study that it is possible to suppress selectively autoreactive B cells by using an artificial antibody chimera that cross-links their inhibitory Fc
RIIb- and antigen-specific receptors. I.v. administration of the chimera to young lupus-prone MRL/lpr mice prevented the appearance of disease-associated auto-antibodies and attenuated for a limited period their kidney disease. Auto-antibodies to nucleosome particles are considered to be the earliest nephritogenic antibody population in the SNF1 mouse lupus model, appearing even before the IgG anti-dsDNA auto-antibodies (19). Pure DNA is not immunogenic, but may be rendered so when coupled to proteins such as histones (31, 32).
The H1'22–42 peptide used in this study has been eluted from MHC class II molecules on antigen-presenting cells from SNF1 mice with lupus and has been shown to stimulate autoimmune Th to augment the production of pathogenic anti-nuclear antibodies by B cells from this mouse strain. It has also been shown that immunization of these animals with the H1'22–42 peptide accelerated their kidney disease (19). Attempts to induce a low-dose tolerance to nucleosomal peptides in SNF1 mice were also made. The s.c. administration once every 2 weeks of 1 mg of the H1'22–42 or of other histone peptides to female SNF1 lupus-prone mice resulted in the suppression of serum anti-dsDNA, anti-nucleosomal, but not of anti-histone IgG levels and increased significantly the survival of the animals. These beneficial effects were shown to be due to the induction of T regulatory cell activity that suppressed disease-associated Th2 (33). In this study, we show that the chimera conjugates suppress the production of anti-Histone 1 antibodies and at the same time affect the production of anti-DNA antibodies. This could be explained by the previously described phenomenon of ‘tolerance spreading’, where the deliberately induced silencing of disease-associated lymphocytes with one specificity results in the suppression of pathological B and T cells with additional specificities (20).
A possible sequence of events is the following—the silencing of the pathological Histone 1-specific cells by the 2.4G2-H1'22–42 chimera results in the decreased presentation of nucleosomal antigens to Th. The result being a weaker antibody response to both nucleosomal components (histones and DNA) (1, 31, 34).
The size of spleens and lymph nodes is significantly decreased in the 2.4G2-H1'22–42 chimera-treated lupus mice. The enlargement of lymphoid organs in MRL/lpr mice is due mainly to the accumulation of T-lymphocytes. Our data show that the chimera binds to B, but not to T-lymphocytes. Both seemingly conflicting findings could be reconciled if one has in mind the often neglected role of disease-associated B-lymphocytes as antigen-presenting cells. The silencing of pathological Histone 1-specific B cells by the 2.4G2-H1'22–42 chimera is expected to bring down the activation state as well as the numbers of T cells recognizing nucleosomes, known to be the primary self-antigen that induces the disease (17).
The observed suppression of the anti-Histone 1 (and to some degree of the anti-DNA) IgG antibody levels and the attenuation of glomerulonephritis were not sufficient to affect the final outcome of the disease. The survival of the MRL/lpr mice was not prolonged by the administration of the H1'22–42 peptide chimera, while it was significantly prolonged in the animals treated with a chimera, made of the same antibody, coupled to the artificial DNA-mimicking peptide (35). This difference points to the stronger pathogenic activity of the anti-dsDNA antibodies in this lupus model.
A chimeric molecule composed of the constant region of a human IgG1 antibody coupled to the cat Fel d1 allergen induced a dose-dependent inhibition of Fel d1-driven IgE-mediated histamine release from cat-allergic donors’ basophiles (36). The cross-linking of the Fc
R1 and the inhibitory FcRIIb receptor was shown to be responsible for the observed result. This chimera would obviously also aggregate Fel d1-binding BCRs with the inhibitory receptors on allergen-specific B-lymphocytes. Its effect on the activity of the latter, however, was not studied.
Chromatin has multiple structurally unrelated epitopes and thus the auto-antibody response to it includes antibodies to DNA, single histones, histone–DNA complexes as well as to higher-order conformational structures (16). As we have already shown (35), it is possible to selectively suppress in vitro as well as in vivo the appearance of IgG anti-dsDNA antibodies by the administration of a chimeric molecule containing an artificial DNA-mimicking peptide (37) coupled to the 2.4G2 mAb. The present study uses a natural lupus target antigen and confirms the hypothesis that hybrid antibodies are able to limit undesirable antibody responses by the same mechanism as IgG-containing immune complexes limit the intensity and duration of IgG antibody production under physiological conditions.
|
Funding |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionFundingReferences |
|---|
Howard Hughes Medical Institute (55000340); Bulgarian National Science Fund (L1304/03); The Swiss National Science Foundation (IB73B0-110719).
|
Abbreviations |
|---|
| BCR, B cell receptor |
| ds, double stranded |
| i.v., intravenously |
| MOG, myelin oligodendrocyte glycoprotein |
| RT, room temperature |
| s.c., subcutaneously |
| SLE, systemic lupus erythematosus |
| SNF1, (SWR x NZB)F1 |
| SpA, superantigen |
| TBS, Tris-buffered saline |
|
Notes |
|---|
Transmitting editor: S. Izui
Received 6 July 2006, accepted 5 November 2007.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionFundingReferences |
|---|
- Shlomchik MJ, Craft JE, Mamula MJ. From T to B and back again: positive feedback in systemic autoimmune disease. Nat. Rev. Immunol. (2001) 1:147.[CrossRef][Medline]
- Chan OTM, Hannum LG, Haberman AM, Madaio MP, Shlomchik MJ. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J. Exp. Med. (1999) 189:1639.
[Abstract/Free Full Text] - Lipsky PE. Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity. Nat. Immunol. (2001) 2:764.[CrossRef][Web of Science][Medline]
- Weide R, Heymanns J, Pandorf A, Koppler H. Successful long-term treatment of systemic lupus erythematosus with rituximab maintenance therapy. Lupus (2003) 12:779.
[Abstract/Free Full Text] - Looney RJ, Anolik J, Sanz I. B-lymphocytes in systemic lupus erythematosus: lessons from therapy targeting B cells. Lupus (2004) 13:381.
[Abstract/Free Full Text] - Gorman C, Leandro M, Isenberg D. B cell depletion in autoimmune disease. Arthritis Res. Ther. (2003) 5:S17.[CrossRef][Medline]
- Looney RJ, Anolik JHA, Campbell D, et al. B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/II dose-escalation trial of rituximab. Arthritis Rheum. (2004) 50:2580.[CrossRef][Web of Science][Medline]
- Yazawa N, Hamaguchi Y, Poe JC, Tedder TF. Immunotherapy using unconjugated CD19 monoclonal antibodies in animal models for B lymphocyte malignancies and autoimmune disease. Proc. Natl Acad. Sci. USA (2005) 102:15178.
[Abstract/Free Full Text] - Murakami M, Yoshioka H, Shirai T, Tsubata T, Honjo T. Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells. Int. Immunol. (1995) 7:877.
[Abstract/Free Full Text] - Viau M, Zouali M. Effect of the B cell superantigen protein A from S. aureus on the early lupus disease of (NZBxNZW) F1 mice. Mol. Immunol. (2005) 42:849.[CrossRef][Web of Science][Medline]
- Patole PS, Zecher D, Pawar RD, Grone H-J, Schlondorff D, Anders H-J. G-rich DNA suppresses systemic lupus. J. Am. Soc. Nephrol. (2005) 16:3273.
[Abstract/Free Full Text] - Reiners KS, Hansen HP, Krussmann A, et al. Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen. Immunology (2004) 112:228.[CrossRef][Web of Science][Medline]
- Zocher M, Baeuerle PA, Dreier T, Iglesias A. Specific depletion of autoreactive B-lymphocytes by a recombinant fusion protein in vitro and in vivo. Int. Immunol. (2003) 15:789.
[Abstract/Free Full Text] - Burny W, Lebrun P, Cosyns JP, Saint-Remy JM. Treatment with dsDNA-anti-dsDNA antibody complexes extends survival, decreases anti-dsDNA antibody production and reduces severity of nephritis in MRLlpr mice. Lupus (1997) 6:4.
[Abstract/Free Full Text] - Kotzin BL. Systemic lupus erythematosus. Cell (1996) 85:303.[CrossRef][Web of Science][Medline]
- Schett G, Smole J, Zimmermann C, et al. The autoimmune response to chromatin antigens in systemic lupus erythematosus: autoantibodies against histone H1 are a highly specific marker for SLE associated with increased disease activity. Lupus (2002) 11:704.
[Abstract/Free Full Text] - Mohan C, Adams S, Stanik V, Datta S. Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus. J. Exp. Med. (1993) 177:1367.
[Abstract/Free Full Text] - Salaman MR, Mawer DPC, Hogarth MB, Seifert MH, Isenberg DA. Counter-proliferative effects of nucleosomal antigens in cultures from lupus patients. Lupus (2001) 10:332.
[Abstract/Free Full Text] - Kaliyaperumal A, Michaels MA, Datta SK. Naturally processed chromatin peptides reveal a major autoepitope that primes pathogenic T and B cells of lupus. J. Immunol. (2002) 168:2530.
[Abstract/Free Full Text] - Kaliyaperumal A, Michaels MA, Datta SK. Antigen-specific therapy of murine lupus nephritis using nucleosomal peptides: tolerance spreading impairs pathogenic function of autoimmune T and B cells. J. Immunol. (1999) 162:5775.
[Abstract/Free Full Text] - Hamaguchi Y, Xiu Y, Komura K, Nimmerjahn F, Tedder TF. Antibody isotype-specific engagement of Fcgamma receptors regulates B lymphocyte depletion during CD20 immunotherapy. J. Exp. Med. (2006) 203:743.
[Abstract/Free Full Text] - Bolland S, Ravetch JV. Spontaneous autoimmune disease in Fc(gamma)RIIB-deficient mice results from strain-specific epistasis. Immunity (2000) 13:277.[CrossRef][Web of Science][Medline]
- Ravetch JV, Lanier LL. Immune inhibitory receptors. Science (2000) 290:84.
[Abstract/Free Full Text] - Fukuyama H, Nimmerjahn F, Ravetch JV. The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat. Immunol. (2005) 6:99.[CrossRef][Web of Science][Medline]
- Legrain P, Sanchez P, Buttin G. Immune response induced by a single or several syngeneic monoclonal antiABPC48 antiidiotypic antibodies: no predominant coexpression of ABPC48 idiotopes. Mol. Immunol. (1985) 22:445.[CrossRef][Web of Science][Medline]
- Mehok AR, Mant CT, Gera L, Stewart J, Hodges RS. Preparative reversed-phase liquid chromatography of peptides. Isocratic two-step elution system for high loads on analytical columns. J. Chromatogr. A (2002) 972:87.[CrossRef][Web of Science][Medline]
- Bauminger S, Wilchek M. The use of carbodiimides in the preparation of immunizing conjugates. Methods Enzymol. (1980) 70:151.[Medline]
- Shapira M, Jibson M, Muller G, Arnon R. Immunity and protection against influenza virus by synthetic peptide corresponding to antigenic sites of hemagglutinin. Proc. Natl Acad. Sci. USA (1984) 81:2461.
[Abstract/Free Full Text] - Djoumerska IK, Tchorbanov AI, Donkova-Petrini VD, SPashov AD, Vassilev TL. Serum IgM, IgG and IgA block by F(ab)`2-dependent mechanism the binding of natural IgG autoantibodies from terapeutic immunoglobulin preparations to self-antigens. Eur. J. Haematol. (2004) 73:1.[Web of Science][Medline]
- Srebreva L, Kachaunova A, Zlatanova J. The occurrence and properties of histone H1 zero in quiescent rabbit tissue. Int. J. Biochem. (1991) 23:189.[CrossRef][Web of Science][Medline]
- Kang H-K, Michaels MA, Berner BR, Datta SK. Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets. J. Immunol. (2005) 174:3247.
[Abstract/Free Full Text] - Treves S, Bajocchi G, Zorzato F, Govoni M, Trotta F. Identification and characterization of a calreticulin-binding nuclear protein as histone (H1), an autoantigen in systemic lupus erythematosus. Lupus (1998) 7:479.
[Abstract/Free Full Text] - Voynova EN, Tchorbanov AI, Todorov TA, Vassilev TL. Breaking of tolerance to native DNA in nonautoimmune mice by immunization with natural protein/DNA complexes. Lupus (2005) 14:543.
[Abstract/Free Full Text] - Steinman L. A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry. Proc. Natl Acad. Sci. USA (1996) 93:2253.
[Abstract/Free Full Text] - Tchorbanov AI, Voynova EN, Mihaylova NM, Todorov TA, Nikolova M, Yomtova VM, Chiang BL, Vassilev TL. Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice. Eur. J. Immunol. (2007) 37:3587.[CrossRef][Medline]
- Zhu D, Kepley CL, Zhang K, Terada T, Yamada T, Saxon A. A chimeric human-cat fusion protein blocks cat-induced allergy. Nat. Med. (2005) 11:446.[CrossRef][Web of Science][Medline]
- Putterman C, Deocharan B, Diamond B. Molecular analysis of the autoantibody response in peptide-induced autoimmunity. J. Immunol. (2000) 164:2542.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||