Regulation of dendritic cells and macrophages by an anti-apoptotic cell natural antibody that suppresses TLR responses and inhibits inflammatory arthritis - PubMed (original) (raw)

. 2009 Jul 15;183(2):1346-59.

doi: 10.4049/jimmunol.0900948. Epub 2009 Jun 29.

Sahil Khanna, Carl S Goodyear, Yong Beom Park, Eyal Raz, Steffen Thiel, Caroline Grönwall, Jaya Vas, David L Boyle, Maripat Corr, Dwight H Kono, Gregg J Silverman

Affiliations

Regulation of dendritic cells and macrophages by an anti-apoptotic cell natural antibody that suppresses TLR responses and inhibits inflammatory arthritis

Yifang Chen et al. J Immunol. 2009.

Abstract

Although natural Abs (NAbs) are present from birth, little is known about what drives their selection and whether they have housekeeping functions. The prototypic T15-NAb, first identified because of its protective role in infection, is representative of a special type of NAb response that specifically recognizes and forms complexes with apoptotic cells and which promotes cell-corpse engulfment by phagocytes. We now show that this T15-NAb IgM-mediated clearance process is dependent on the recruitment of C1q and mannose-binding lectin, which have known immune modulatory activities that also provide "eat me" signals for enhancing phagocytosis. Further investigation revealed that the addition of T15-NAb significantly suppressed in vitro LPS-induced TNF-alpha and IL-6 secretion by the macrophage-like cell line, RAW264.7, as well as TLR3-, TLR4-, TLR7-, and TLR9-induced maturation and secretion of a range of proinflammatory cytokines and chemokines by bone marrow-derived conventional dendritic cells. Significantly, high doses of this B-1 cell produced NAb also suppressed in vivo TLR-induced proinflammatory responses. Although infusions of apoptotic cells also suppressed such in vivo inflammatory responses and this effect was associated with the induction of high levels of IgM antiapoptotic cell Abs, apoptotic cell treatment was not effective at suppressing such TLR responses in B cell-deficient mice. Moreover, infusions of T15-NAb also efficiently inhibited both collagen-induced arthritis and anti-collagen II Ab-mediated arthritis. These studies identify and characterize a previously unknown regulatory circuit by which a NAb product of innate-like B cells aids homeostasis by control of fundamental inflammatory pathways.

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Figures

Figure 1

Figure 1. T15-IgM NAb enhances deposition of C1q and MBL on apoptotic cells and increases their in vivo phagocytic clearance by peritoneal Mφ

(A) To assess for C1q deposition, etoposide-treated apoptotic thymocytes were incubated in 50% muMT sera in saline or with monoclonal IgM (20 ug/ml), then washed and stained with 7AAD (to assess membrane integrity) and anti-murine C1q or isotype control, as indicated. While labeled Annexin V was used to document apoptosis, it was otherwise omitted to avoid interference with C1q-binding. By gating on early apoptotic cells (i.e., 7AAD−), which are indicated by the arrows, addition of T15 IgM was shown to increase more than 3-fold the level of C1q deposition from Ig-deficient sera, compared to saline or IgM isotype control. At bottom, control studies demonstrated no significant signal (left) on early apoptotic cells with the isotype control detection reagent, (middle) or with early apoptotic cells without MuMT sera but with T15 IgM (no sera), or (right) for C1q deposition onto freshly isolated live cells in the presence of T15-NAb. (B) ELISA studies show binding of IgM with a biotinylated detection reagent to different precoated antigens, which are listed at bottom. With a precoat of PC-albumin, T15-IgM (at 2 ug/ml) displays specific MBL- and antigenic-binding, but not to albumin alone. Specific MBL binding is blocked by mannose or N-acetylglucosamine (NAcGlu) but not by N-acetyl galactosamine (NAcGal) at 20 mM. Binding also requires CaCl2, and is absent in 10mM EDTA-containing buffer. (C) Etoposide-treated or γ-irradiated apoptotic thymocytes were incubated with human recombinant MBL in the absence or the presence of purified monoclonal IgM, then stained with labeled anti-human MBL. At top, binding is shown after gating on early (7AAD-Annexin V+) apoptotic cells. At bottom left, control studies demonstrated no significant signal on apoptotic cells with the biotinylated isotype-control detection reagent. (D) To assess for the capacity to recruit murine MBL from sera, etoposide-treated thymocytes were incubated with MuMT sera in the presence of saline or purified monoclonal IgM, as indicated. Cells were stained for IgM, and with labeled anti-mouse MBL A and MBL C. In control studies (bottom), only background reactivity is seen after incubation of apoptotic cells sera and T15-IgM, with staining with an isotype control reagent (left). Incubation without sera yielded background signal with anti-MBL A+C detection, while MBL deposition was greatly reduced by the addition of mannose (50 uM) to sera and T15-IgM. T15-IgM and sera incubation did not result in MBL deposition on healthy cells (right). (E) In vivo Mφ mediated apoptotic clearance was evaluated, in three independent experiments, as indicated (solid triangle, open circles and solid circles), with 2–4 B-cell deficient mice in each group. Mice received either T15-IgM, isotype control IgM or saline treatment before instillation of apoptotic thymocytes (total N=9–10/group). Values for each mouse represent the phagocytic index after 10 min, (i.e., proportion of recovered peritoneal Mφ that had engulfed labeled apoptotic thymocytes) (F) Cytospin preps show NAb enhanced phagocytic engulfment of apoptotic cells by peritoneal Mφ from B-cell deficient mice that received IgM or saline. After 16 hr, thymocytes labeled with SNARF-1 fluorochrome (red) were instilled i.p., with sacrifice 10 min later. Mφ were detected by F4/80 FITC (green). At top, treatment with specific IgM treatment is indicated, with either apoptotic or healthy freshly isolated thymocytes, as indicated below. Results are representative of three or more independent experiments.

Figure 2

Figure 2. T15-NAb inhibits LPS induced secretion of TNF-a and IL-6 by the RAW264.7 macrophage cell line

RAW 264.7 murine macrophage cells were stimulated overnight without or with LPS (100 ng/ml), and supernatants were assayed for (A) TNF-α and (B) IL-6. Values represent mean +/− SEM or 3 or more replicate cultures. Representative of 4 experiments.

Figure 3

Figure 3. T15-NAb enhances in vitro phagocytosis of apoptotic thymocytes by conventional DC

Bone marrow derived purified DC were cultured with equal numbers of CFSE-labeled apoptotic cells in serum-free media for 1 hr. Cultures were supplemented with IgM, purified human C1q, recombinant human MBL, or Ig-deficient murine sera as indicated. For analysis, cell fragments were removed based on forward scatter, which enabled the discrimination of the proportion of DCs that have engulfed apoptotic cells (ACs). (A) Cultures included limiting concentrations of T15 or isotype control IgM (5 ug/ml), with saturating amounts of MBL (40 ug/ml), as indicated. High levels of AC phagocytosis requires co-culture with both T15-NAb and MBL, while the level of phagocytosis is inhibited by preincubation with PC-BSA (50ug/ml) but not by control protein conjugate (ABA-BSA). (B) PC significantly inhibited T15-NAb mediated enhancement of AC phagocytosis, as shown in data compiled from replicate cultures. (C) MBL is responsible for large dose-dependent increases in AC engulfment increases in the presence of T15-IgM (at 20 ug/ml), with significantly less effects in cultures without IgM or with isotype control (20 ug/ml). (D) Replicate cultures demonstrate equivalent significant T15-IgM mediated increases with addition of Ig-deficient sera, while C1q also conveys significant but smaller increases in T15-NAb dependent AC phagocytosis, as recently reported (18).

Figure 4

Figure 4. T15-NAb inhibits in vitro TLR induced maturation of conventional DC

Bone marrow mononuclear cells were cultured with GM-CSF and IL-4 for 5 days, then purified with anti-CD11c beads prior to overnight culture in serum-free media. (A) LPS induces maturation of DC, based on increased CD80 and IL-12p40 expression, which is inhibited by T15 IgM (20 μg/ml) in cultures supplemented MBL (20 μg/ml) and C1q (80 μg/ml). DC are shown to be heterogeneous in their level of maturation, even in the absence of LPS or poly I:C. Based on scattergram analysis, these overnight cultures contained 10–18% dead cells and fragments. (B) T15-NAb, in the presence of MBL and C1q, inhibits LPS induced DC maturation. Values for CD80− IL-12p40low DC, as gated in panel A, is shown from four or more replicate cultures (mean+/− SEM). (C) T15-NAb, in the presence of MBL and C1q, inhibits poly I:C induced DC maturation. Data from four or more replicate cultures are shown. Open bars are without IgM, dark bars are with Isotype control, and vertical strips are with T15 IgM at 20 ug/ml. Significance as indicated: * P<0.05; ** ≤ 0.01; *** ≤0.0001; **** ≤0.0003. Representative of three independent experiments.

Figure 5

Figure 5. T15-NAb treatment blunts in vitro DC responses to TLR agonists

(A) CD11c+ selected myeloid DC were cultured in replicate with agonists for TLR3 (poly I:C), TLR4 (LPS), TLR7 (imiquimod), or TLR9 (PT CpG ODN1018), without or with T15-IgM or isotype control at indicated concentrations. (A) Histograms of MHC II and CD40 on DC after culture without or with stimulant (indicated above panel) are depicted. Mean fluorescence intensity is listed without and with IgM, at indicated concentrations. (B) Supernatants from these overnight cultures of conventional bone-marrow derived DC were assessed for levels of pro-inflammatory cytokines and chemokines, which were determined from standard curves (mean+/−SEM). Results are shown without (none) or with stimulants (poly I:C, pIC; imiquimod, imiq) without or with T15-IgM, isotype IgM control, at 10 ug/ml. The isotype control was associated with minor inhibition of MCP-1. (C) Transcript levels were determined by real time PCR for murine BM-derived CD11c+ DC, under indicated cultured conditions over time (min). DC were preincubated with T15-NAb or isotype control before time “0” sampling, then LPS was added. Amplification for TGFβ is β1 isoform-specific. Results are representative of three or more independent experiments.

Figure 6

Figure 6. In vivo T15-IgM treatment blunts responses to TLR agonists

(A) Groups of adult C57BL/6 mice received saline, IgM control or T15-IgM, then were challenged with agonists for TLR3 (poly I:C), TLR4 (LPS), TLR7 (SM-360320)(27), TLR9 (CpG ODN1018). After 18hr, splenic Mφ and CD11chi DC were evaluated, based on indicated gates (left panels). Representative histograms and mean fluorescence indices (MFI) are depicted for: top value from gray shaded area from naïve mouse; saline pretreatment then TLR agonist challenge dark solid line; isotype control followed by TLR agonist challenge from thick gray dashed line; bottom, T15-NAb followed by TLR agonist, black dashed line. Compared to naïve mice, challenge with TLR agonist induces high expression levels of MHC II and CD86, but was inhibited by T15-NAb. (B) T15-IgM or apoptotic-cell treatments inhibited poly I:C induced activation of splenic Mφ (F4/80+) and myeloid DC (CD11chi) from C57BL/6 mice. Groups received buffer alone (PBS), necrotic (Necr.) thymocytes, apoptotic (Apop.) thymocytes, isotype control (Iso IgM), T15 or the indicated combination. Results are shown for individual mice at 18 hr after challenge. Horizontal bars depict mean values for each group. There were no significant differences in the representation of splenic F4/80+ Mφ and CD11chi DC between different treatment groups. (C) T15-IgM or apoptotic-cell treatments inhibits poly I:C induction of serum levels of pro-inflammatory cytokines and chemokines in C57BL/6 mice. P values from two-tailed t test are shown. Results were pooled from three or more independent experiments. (D) Representative flow cytometric analyses after gating on early apoptotic thymocytes (i.e., Annexin V+ 7AAD−) of IgM and MBL from sera of naïve adult C57BL/6, or mice that received three weekly apoptotic cell (AC) treatments, with level of binding shown to be dose-dependent based on the % sera. PC-BSA incubation greatly reduced the level of MBL deposition. Representative of three independent studies.

Figure 7

Figure 7. B-cell deficient mice are not protected by apoptotic-cell treatments

T15-IgM but not apoptotic-cell treatments inhibits poly I:C-induced activation of splenic Mφ and myeloid DC in B-cell deficient (muMT) mice. Results are for MFI from flow cytometry studies, as shown in Figures 6A&B. Results are for individual mice are the pooled from two independent experiments.

Figure 8

Figure 8. T15-NAb protects from inflammatory arthritis

(A) DBA/1 mice were immunized with CII and boosted on day 20. T15-NAb at 2 mg/dose, isotype control, apoptotic thymocytes or necrotic cells (2.5 × 107) in saline, or saline alone were administered weekly. T15-NAb and apoptotic-cell treatments significantly reduced clinical arthritis joint scores compared to control treatments (isotype control, saline and necrotic cells) (P<0.001 by Bonferroni test). The isotype control group was not significantly different than saline-treated group. Data are pooled from two independent studies with separate treatment and control groups of four mice (total N=8). Depicted are mean values+/−SEM. (B) Protective T15-NAb reduced inflammatory cellular infiltrates in CIA. Compared to isotype control treatment at left, T15-IgM anti-PC NAb significantly reduced cartilage and bone destruction (arrowhead), and greatly reduced level of cellular infiltrates (arrow)(40Xmag). Below, knees from control-treated mice had progressive pathologic changes of compromised articular cartilage that is shown with safranin O (bright orange), while T15-IgM provided protection from cellular infiltrates, and cartilage and bone destruction. (C) Histologic arthritis scores are depicted for CIA treatment study, with values derived as previously described (28). (D) To induce autoantibody-mediated arthritis, BALB/c mice received a commercial cocktail of anti-CII antibodies, and data represent sequential measurements from two independent studies with separate treatment and control groups of four mice (total N=8). Weekly T15-NAb infusions significantly reduced arthritis based on clinical scores of joint scores, compared to saline or isotype control-treated mice, with P<0.0022 at the peak d14 response. Depicted are mean values +/− SD.

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