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Keywords:

  • Autoimmune disease;
  • B cells;
  • CD22;
  • Inflammation;
  • IVIg

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References

Intravenous immunoglobulins (IVIgs) efficiently suppress a variety of autoimmune diseases. Over the past few years several potential mechanisms underlying this antiinflammatory activity have become apparent. Among these, terminal sialic acid residues in the sugar moiety of the immunoglobulin G constant fragment have been shown to be critical for the antiinflammatory activity of IVIgs in models of rheumatoid arthritis and immunothrombocytopenia (ITP). More recently, B cells and the sialic acid-binding protein CD22 were suggested to be involved in this IVIg-dependent immunomodulatory pathway. To study whether B cells are directly involved in IVIg-mediated suppression of acute autoimmune diseases, we tested the activity of IVIgs in mice deficient in B cells or CD22. We show that neither B cells nor CD22 are critical for the immediate antiinflammatory activity of IVIgs in mouse models of rheumatoid arthritis and ITP.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References

Since the first demonstration that the infusion of high doses of immunoglobulin G (IgG) preparations pooled from several thousands of donors can interfere with autoantibody-mediated platelet depletion by Imbach et al. [1] nearly 30 years ago, the use of IVIgs as an antiinflammatory therapy has dramatically expanded [2-5]. Besides the few licensed indications for high dose IgG therapy in autoimmune diseases such as immunothrombocytopenia (ITP), Guillain-Barre syndrome, Kawasaki's disease, and chronic inflammatory demyelinating polyneuropathy many other acute or chronic inflammatory diseases have been treated with IVIgs. In human ITP patients and in mouse models of ITP, nephrotoxic nephritis, and rheumatoid arthritis (RA) it was shown that the IgG Fc-fragment is critical for the antiinflammatory activity of IVIgs [6-11]. Moreover, the sugar moiety attached to the IgG constant fragment and especially terminal sialic acid residues were shown to be essential for the therapeutic activity of IVIg preparations in mouse models of ITP and RA [6, 7, 11-13]. As human and mouse IgG rich in terminal sialic acid residues have a reduced affinity for the family of canonical Fcγ-receptors (FcγRs) it was suggested that other IgG glycosylation-specific Fc-receptors may exist [11, 14]. Indeed, SignR1 and human DC-SIGN were identified to be able to directly recognize sialic acid rich IgG glycovariants and blocking SignR1 function interfered with the antiinflammatory activity of IVIgs in mouse models of ITP and RA [12, 13]. Apart from SignR1, mice deficient in FcγRIIB no longer responded to IVIg treatment in models of ITP, RA, and nephrotoxic nephritis, although more recently mouse strain dependent differences with respect to the absolute requirement of FcγRIIB in IVIg-dependent amelioration of ITP were noted [10, 11, 15-17]. Moreover, an increased expression of the inhibitory FcγRIIB on myeloid cells and B cells was observed in mice and human chronic inflammatory demyelinating polyneuropathy patients following IVIg infusion, suggesting that the threshold for cell activation and induction of proinflammatory effector functions becomes reset upon high dose IgG infusions [4, 8, 10, 11, 16, 18]. While the influence of IVIg infusion on innate immune effector cells is well described it has become clear only recently that cells of the adaptive immune system including B cells are influenced by IVIg therapy, demonstrated by the upregulation of FcγRIIB on mouse and human B cells for example [18, 19]. Moreover, several studies found that TLR9-dependent B-cell responses are modulated via IVIgs and B cells are well known to be able to modulate immune responses by the release of antiinflammatory cytokines including IL-10 [20-23]. As B cells do not express SignR1 or DC-Sign it seems likely that other cell surface receptors on B cells may have the capacity to recognize sialic acid rich IgG glycovariants. An obvious candidate protein expressed selectively on B cells able to recognize sialic acid residues is CD22, which is a negative regulator of B-cell activation [24]. Indeed, a recent study performed with human B cells in vitro suggested that sialic acid rich IgG may bind to B cells via CD22 and result in a reduced survival [25]. To study if B cells or CD22 contribute to the well-established antiinflammatory activity of IVIgs in mouse models of ITP and RA, we made use of mouse strains deficient in B cells or CD22. We show that despite the capacity of B cells to directly bind to IVIgs in vivo, this is not critical for the suppression of these two types of autoantibody-mediated autoimmune diseases. Thus, while IVIg-dependent modulation of B-cell activation or survival may have some delayed impact on the development of autoimmune diseases it is not required for its immediate immunosuppressive activity in vivo.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References

Binding of IVIg to cells in the peripheral blood

Several studies performed with human peripheral blood cells reported that IVIg binds to different human cell populations in vitro and can affect their survival, although other studies have not seen such effects [25-29]. To study if these effects are reproducible in vivo we first analysed binding of IVIg to different cell populations in the peripheral blood of mice. As shown in Figure 1, we could confirm these findings and demonstrate that IVIgs can bind to B cells, CD11b+ cells and T cells upon intravenous injection (Fig. 1A–C, left and upper panels). As CD22 has been suggested to be one of the molecules critical for IVIg binding to B cells, we injected CD22 knockout mice with IVIgs and performed the same binding experiment as for wild-type mice. As shown in Figure 1A–C, injection of IVIgs into CD22-deficient mice did not result in a reduction of binding to any of these cell types (Fig. 1A–C middle panels). Similarly, injection of IVIgs or neuraminidase-treated IVIg preparations devoid of terminal sialic acid residues showed an unaltered-binding pattern, suggesting that neither CD22 nor its potential ligand (terminal sialic acid residues) are essential for IVIg binding to B cells, myeloid cells, and T cells (Fig. 1B, upper and lower panel). The capacity of IVIg to bind to T cells also suggests a minor role of FcγRs in this process, as T cells do not express this family of molecules. Of note, none of these cell types expresses SIGNR1, which is restricted to splenic and lymph node macrophage subpopulations, suggesting that other, as yet unknown cell surface receptors have the capacity to recognize human IVIgs.

image

Figure 1. IVIg binding to different cell populations in the peripheral blood in vivo. Analysis of human IgG binding to different cell subsets in the blood 1 h after intraperitoneal application of 1 g/kg IVIg or neuraminidase digested IVIg (Neur.IVIg) in C57Bl/6 and CD22−/− mice by (A and B) FACS analysis and by (C) immunofluorescence with antibodies directed against B cells (green), T cells (blue) and human IgG (magenta). The quantification of IVIg binding (B) to the indicated cell populations was performed by calculating the increase in the median fluorescence intensity (Δ MFI) between cells before (gray histograms in (A)) and 1 h after (black histograms in (A)) IVIg injection. Data are shown as mean + SD and are representative of three independent experiments.

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Impact of IVIg treatment on B cells and the cytokine signature of the blood

We next set out to determine whether IVIg treatment affects B-cell numbers in the blood in vivo. Peripheral blood of C57Bl/6 mice was withdrawn before and 24 h after application of IVIg or PBS and absolute cell numbers were determined. Apart from natural fluctuations in absolute cell numbers of B cells and CD11b-positive cells, which could also be observed in PBS-treated mice, cell numbers did not differ between the two experimental groups, suggesting that at least in vivo IVIg does not affect B-cell viability, which is consistent with some but not all studies performed before with human B cells in vitro (Fig. 2) [25-27]. Besides a B-cell depleting mechanism, it remained possible that IVIgs could trigger the release of antiinflammatory cytokines by B cells such as IL-10 that may result in immediate antiinflammatory effects [20, 21]. To investigate this, we first analyzed the serum cytokine status of IVIg and PBS-treated C57Bl/6 mice. Despite a slight but nonsignificant increase of most cytokines under investigation in IVIg-treated mice a high dose infusion of human IgG molecules did not cause a systemic release of cytokines (Fig. 3). Interestingly, we noted a small increase in serum ILx02010;4 levels, which has been described to be involved in an IVIg triggered TH2-dependent antiinflammatory pathway in a model of RA [6]. As this ILx02010;4 induction was at the detection limit and not present in all mice of the group, however, this value did not reach statistical significance. Due to the virtually unchanged cytokine milieu we refrained from studying cytokine release by individual cell subsets in further detail.

image

Figure 2. Maintenance of B-cell number after IVIg treatment. Shown are the absolute numbers of peripheral blood B cells and CD11b+ cells of C57Bl/6 mice before and 24 h after injection of either IVIg or PBS. Data are shown as means + SD and show one out of three independent experiments.

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image

Figure 3. Cytokine pattern after IVIg treatment. Shown are the serum levels of the indicated cytokines in C57Bl/6 mice 2 h after injection of either PBS or IVIg (1 g/kg). Data are shown as mean + SD of 5–6 mice per group and are representative of one out of two independent experiments. ns: no significant difference.

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The role of B cells and CD22 in IVIg-mediated suppression of ITP and RA

While our previous results argue against a role of some major pro- and antiinflammatory cytokines in the activity of IVIg, it remained possible that B cells participate by other means in suppressing autoantibody-dependent effector responses. Therefore, we studied IVIg activity in B-cell deficient and in CD22 knockout mice in a model of autoantibody-induced ITP in which IVIg-mediated suppression of autoimmune diseases is abrogated if terminal sialic acid residues are removed from the IVIg preparation [13] (Fig. 4A–C). As B-cell deficient mouse model systems we used either μMT mice, lacking mature B-cell populations, or mice treated with a CD20-specific antibody to deplete B cells as described before [30, 31] (Fig. 4D–E). The result of these experiments was that IVIg was fully active and suppressed the antiplatelet antibody induced thrombocytopenia independently of B cells or CD22. To show that IVIg activity was still dependent on terminal sialic acid residues in the absence of B cells or CD22 we repeated these experiments with neuraminidase-treated IVIg to deplete terminal sialic acid residues. As shown in Figure 4F–H, IVIg activity was abrogated in the absence of terminal sialic acid residues in B-cell depleted, μMT knockout and in CD22 knockout mice. To further extend our results to other model systems we used the K/BxN serum transfer model [32]. As in the ITP model, IVIg-mediated suppression of joint inflammation is critically dependent on terminal sialic acid residues in this model system as well [11]. Consistent with the previous results, IVIg-mediated suppression of joint inflammation was not changed in CD22 and μMT knockout mice compared with the C57Bl/6 controls (Fig. 5). This suggests that at least in these two in vivo model systems, in which a role of terminal sialic acid residues in IVIg activity has been proven, neither B cells nor CD22 are involved in the sialic acid-dependent antiinflammatory pathway.

image

Figure 4. Role of B cells and CD22 in IVIg-dependent suppression of ITP. Mice received either 1 g/kg IVIg or PBS 2 h before induction of ITP by injection of 0.2 μg/g of the platelet-specific antibody 6A6-IgG2a. Shown are the residual platelet counts of (A) C57Bl/6 mice, (B) μMT knockout mice, (C) CD22-deficient mice, and (D) B-cell depleted mice 4 h after ITP induction. (E) Shown are representative FACS analysis experiments identifying B-cell counts in the peripheral blood of C57Bl/6 mice before and 72 h after injection of 100 μg of an CD20-specific IgG2a antibody (α-CD20, upper panel) or PBS (lower panel). (F) B-cell depleted, (G) μMT, or (H) CD22 knockout mice received either PBS, 1 g/kg IVIg or 1 g/kg neuraminidase treated IVIg (Neur.IVIg) 2 h before induction of ITP via injection of 0.2 μg/g of the platelet-specific antibody 6A6-IgG2a. Depicted are the residual platelet counts 4 h after induction of ITP. Data are shown as means + SD and are representative for 2–3 independent experiments with 5–6 mice per group. Statistical analysis was performed with the Wilcoxon-matched pairs test (nonparametric). * p < 0.05; ** p < 0.01; *** p < 0.001; ns: no significant difference.

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image

Figure 5. Role of B cells and CD22 in IVIg-dependent suppression of RA. Shown is the development of clinical signs of arthritis in (A) C57Bl/6, (B) CD22 knockout, and (C) μMT knockout mice after injection with arthritis inducing sera from K/BxN mice. Within each strain a group mice was injected either with PBS or with 1 g/kg IVIg 2 h before arthritis induction. (D) Quantification of the clinical arthritis score 10 days after injection of arthritogenic serum with or without pretreatment with IVIg. Data are shown as mean ± SD are representative of three independent experiments. Statistical analysis was performed with the Wilcoxon-matched pairs test (nonparametric). * p < 0.05; ** p < 0.01.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References

Several different cell populations and mechanisms of activity have been shown to be operative in IVIg-mediated suppression of a wide variety of autoimmune diseases [2-5, 33-36]. Among these different pathways, IVIg glycoforms rich in terminal sialic acid residues have been suggested to be critical for IVIg activity in several but not all mouse model systems [6, 7, 11-13, 37]. A variety of studies suggest that myeloid cells are modulated via sialic acid rich IgG glycovariants by interaction with cell surface receptors such as SignR1 in mice and DC-Sign in humans, providing a plausible explanation for the reduced effector cell-dependent tissue inflammation [6, 8, 11, 12]. Whereas it is known since a long time that B-cell development and responses may be modulated via the presence of anti-idiotypic antibodies, which are able to recognize the B-cell receptor in the IVIg preparation, it was noted only recently that FcγRIIB expression on B cells becomes upregulated by IVIg infusion in mice and humans [18, 19, 38, 39]. As B cells, besides their obvious role in being the source of autoantibody production, can also have strong direct antiinflammatory and immunomodulatory functions, we set out to test if they play a direct role in IVIg-mediated suppression of ITP and RA. Indeed, a previous study showed that B cells may have the capacity to recognize sialic acid rich IgG via CD22 [25]. Moreover, IVIg was suggested to induce apoptosis in primary B cells and in different B cells lines most dramatically 24 h after coincubation with IVIg in vitro [25]. In vivo, however, we did not observe a reduction in peripheral B-cell counts, despite the capacity of IVIg to bind to B cells and other cell types in the blood consistent with other studies showing that the proliferation of human B cells is not influenced by IVIg in vitro. More importantly, this binding was independent of the presence of terminal sialic acid residues and did not require FcγRs or CD22 suggesting that other as yet unidentified molecules on mouse peripheral blood cells can recognize IVIgs. Apart from the induction of apoptosis, IVIgs might be able to stimulate B cells to dampen an ongoing innate immune response via release of ILx02010;10 as shown to be critical for dampening EAE [20, 21]. However, IVIg infusion did not result in a dramatic systemic cytokine release, which is consistent with previous studies showing that IVIg activity was preserved in mouse strains deficient in a variety of cytokines or functional cytokine receptors in different models of mouse ITP [35, 40]. Finally, neither B cells nor CD22 were involved in IVIg-dependent suppression of autoantibody-induced ITP and RA, in which terminal sialic acid residues were shown to be absolutely required for the antiinflammatory activity. It should be noted, that the previous study showing an involvement of CD22 in binding to sialic acid rich IgG glycoforms used intact IVIg preparations to enrich for IgG fractions rich in terminal sialic acid residues [25]. As about 20% of serum IgG can have an additional and more accessible sugar moiety attached to the IgG variable fragment it seems likely that predominantly F(ab)2 sialylated IgG glycoforms were enriched, which may bind to CD22 but do not have an antiinflammatory activity as described by others [10, 11, 41-43]. This may explain at least in part the lack of a role of CD22 in the immunosuppressive function of the immediate antiinflammatory activity of IVIgs in vivo.

Nonetheless, human and mouse B cells have clearly been shown to respond to IVIg therapy by upregulation of FcγRIIB expression for example. As FcγRIIB is a critical checkpoint of humoral tolerance in mice and humans this may indicate that IVIgs might rather have a delayed effect on B cell and antibody responses by raising the threshold for B-cell activation. Moreover, isolated cross-linking of FcγRIIB by immune complexes on plasma cells has been suggested to induce apoptosis and open new niches for de novo generated plasma cells [44-46]. Moreover, IVIg was shown to modulate TLR9-dependent activation of B cells and interfere with B-cell dependent antigen presentation [23, 47]. Therefore, future studies focusing on IVIg-mediated modulation of B-cell development and antibody responses will be necessary to understand if IVIg, and especially the sialic acid rich fraction, affects humoral immune responses in vivo. Supporting this notion, previous reports suggested that the anti-idiotype specific fraction in the IVIg preparation can affect B-cell development and function [39].

In general, care should be taken when trying to transfer data from mouse model systems directly to humans. Although some IVIg-mediated effects, such as the upregulation of FcγRIIB on myeloid cells and B cells can be observed in mice and humans, other players involved in the antiinflammatory activity are quite different. This may be exemplified by the cellular expression pattern of the molecules critical for recognition of sialic acid rich IgG in mice and humans. Whereas SIGNR1 is present on macrophage subpopulations in mouse lymphoid tissue, the human orthologue DC-SIGN shows a more DC-restricted expression pattern [6, 12, 48]. Along the same lines, the genetic background of different mouse strains critically affects the penetrance of autoimmunity, which may explain why FcγRIIB deletion reduced the antiinflammatory activity of IVIg in some but not all strains of mice [8, 10, 16, 17, 49-51]. Ultimately, integrating our knowledge from both, mouse in vivo and human in vitro studies, may allow us to find common denominators of IVIg-mediated suppression of autoimmune pathology.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References

Animals

Female mice at 8–16 weeks of age on the C57Bl/6 background obtained from Janvier (Le Genest-Saint-Isle, France) were used in all experiments. CD22 knockout mice were provided by Lars Nitschke. μMT knockout mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). KRN TCR transgenic mice on a C57Bl/6 background (K/B) were gifts from D. Mathis and C. Benoist (Harvard Medical School, Boston, USA) and were bred with NOD mice to generate K/BxN mice. All mice were maintained under specific pathogen-free conditions and according to the guidelines of the National Institute of Health and the legal requirements in Germany and the United States.

Antibodies and reagents

6A6-IgG2a antibodies were produced by transient transfection of 293T cells as described [52]. Anti-B220/CD45R, anti-CD19, anti-human-IgG, and anti-TCRβ antibodies were purchased from BD Pharmingen. Anti-CD11b and anti-CD45 antibodies were purchased from Biolegend. Neuraminidase, an Acetyl-neuraminyl hydrolase (Sialidase), catalyzes the hydrolysis of α2–3, α2–6, and α2–8 linked N-acetyl-neuraminic acid residues from glycoproteins and oligosaccharides, was purchased from New England Biolabs (Frankfurt, Germany). Neuraminidase digestion of IVIg was performed as suggested by the manufacturer.

Flow cytometry

Flow cytometry analysis was done on a FACS Canto II (BD Bioscience) with single cell suspensions of peripheral blood. Blood was obtained from the retroorbital plexus and erythrocytes were lysed. To reduce unspecific binding to Fc receptors cells were incubated on ice for at least 10 min with Fc block (clone 2.4G2, 0.5 μg per sample). Cells were washed and incubated on ice for at least 20 min with combinations of the following antibodies: anti-B220/CD45R (clone RA3–6B2; BD Pharmingen), anti-CD19 (clone 1D3; BD Pharmingen), anti-CD11b (clone M1/70; Biolegend), anti-CD45 (clone 30-F11; Biolegend), anti-TCRβ (clone H57–597; BD Pharmingen), and anti-human-IgG (clone G18–145; BD Pharmingen). Analysis was restricted to viable cells, which were identified by exclusion of the nucleic acid-binding dye DAPI. Data acquisition and analysis was performed with FACS Diva software (BD Bioscience).

Immunofluorescence

For immunofluorescence of blood lymphocytes, blood from IVIg or PBS pretreated mice was obtained from the retroorbital plexus and erythrocytes were lysed. Cells were resuspended in 150 μL PBS, placed into the Cellspin I Centrifuge (THARMAC) and spinned on microscope slides for 10 min at 12,000 rpm, dried and fixed for 10 min with 3% PFA. Slides were stained with combinations of the following antibodies: anti-B220/CD45R, anti-TCRβ, and anti-human-IgG. After incubation, the excess of fluorescent dye was removed by multiple washing steps with PBS. Microscope slides were examined and analyzed by using an Axiovert 200 M fluorescence microscope (Carl Zeiss). All exposure times and contrast adjustments were kept identical between different samples.

B-cell depletion

For depletion of B cells, mice were injected i.v. with 100 μg of a CD20-specific antibody as described before [30]. B-cell counts were determined before, 24 and 72 h after anti-CD20 antibody injection by FACS analysis. B-cell counts before antibody injection were set to 100%.

ITP induction and therapeutic intervention

ITP was induced by injection of 0.2 μg/g 6A6-IgG2a antibody as described previously [53]. Platelet counts were determined before and 4 h after antibody injection of a 1:4 dilution in PBS and 5% BSA in an Advia 120 hematology system (Bayer). Platelet counts before antibody injection were set to 100%. Either 1 g/kg IVIg (Kiovig, Bayer), neuraminidase-treated IVIg (as described in [11]) or PBS was injected intraperitoneally 2 h prior to ITP induction.

Serum transfer arthritis and therapeutic intervention

Over several weeks collected sera of K/BxN mice were pooled and frozen in aliquots. Arthritis was induced by one injection of 14 μL/g of K/BxN serum. Either 1 g/kg IVIg (Kiovig, Bayer) or PBS was injected 2 h prior to arthritis induction. Arthritis was scored by clinical examination and the index of all four paws was added: 0 (unaffected), 1 (swelling of one joint), 2 (swelling of more than one joint), 3 (severe swelling of the entire paw) as described [11].

Statistical analysis

Statistical differences of clinical scores were calculated with Mann–Whitney-U test. All statistical differences of ITP experiments were determined with Wilcoxon-matched pairs test. A p value < 0.5 was considered significant.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References

We are grateful to Georg Schett for IVIgs, to Jeffrey Ravetch for mice and reagents and to Melissa Woigk, Heike Albert, and Heike Danzer for expert technical assistance. This study was supported by a grant from the Bavarian Genome Research network (BayGene to F.N.) and the Bill and Melinda Gates foundation (OPP1032817 with F.N. as participating investigator).

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References

The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. ACKNOWLEDGMENTS
  8. Conflict of interest
  9. References
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Abbreviations
ITP

immunothrombocytopenia

IVIg

intravenous immunoglobulin

RA

rheumatoid arthritis