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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

Background : Adalimumab, a fully human monoclonal antibody to tumour necrosis factor, was recently introduced for therapy of Crohn's disease.

Aim : Since induction of apoptosis of inflammatory cells is thought to be an important mechanism of action of the antitumour necrosis factor monoclonal antibody infliximab, we studied the induction of apoptosis of activated peripheral blood monocytes by adalimumab.

Method : Apoptosis was analysed at the levels of the cell membrane, mitochondria and DNA by flow cytometry.

Results: We found that both adalimumab and infliximab induced apoptosis in cultured monocytes, while etanercept did not. Apoptosis induction was caspase-dependent and detectable already after 2 h. The production of interleukin-10 and interleukin-12 by monocytes was down-regulated significantly by adalimumab and infliximab but not by etanercept, while levels of soluble tumour necrosis factor in monocyte cultures were down-regulated by all three reagents.

Conclusions : These data show that both adalimumab and infliximab affect monocyte cytokine production and induce apoptosis of activated monocytes. Our findings will have to be further correlated to therapeutic efficacy of these antitumour necrosis factor reagents.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

Tumour necrosis factor (TNF)-α is a cytokine, which exerts its activity as either the cell surface-associated 26-kDa prepeptide [transmembrane TNF-α (tmTNF-α)] or as the cleavage product, the 17-kDa secreted protein (soluble TNF-α) generated by TNF-α-converting enzyme.1 The proinflammatory effects of TNF-α are important in the pathogenesis of inflammatory bowel disease (IBD).2 A wide array of biological agents has been designed to inhibit TNF-α,3 especially the antihuman TNF-α monoclonal antibodies (mAb) infliximab (chimaeric) and adalimumab (fully humanized); and soluble TNF receptor fusion constructs: p75 subunit (etanercept) and p55 subunit (onercept). Surprisingly, results of treatment with different neutralizing reagents in Crohn's disease (CD) are divergent. Infliximab, the chimaeric immunoglobulin (Ig)G1, has been shown to have high efficacy for the treatment of luminal and fistulizing CD as well as for the control of extraintestinal manifestations of IBD.4–6 Adalimumab, whose human nature is expected to be associated with less immunogenicity than a chimaeric one, was found effective in CD in a recent update report.7 CDP-571 (an IgG4 isotype variant of infliximab) and CDP-870 (an F(ab’)2 fragment of infliximab) are also although somewhat less effective.8–12 On the contrary, the result of treatment with etanercept was disappointing.13 The importance of apoptosis induction to obtain disease remission was suggested by several groups. The exact mechanism is unclear, but infliximab is known to bind both soluble and membrane-bound TNF, and to kill TNF-expressing cells.14 Lugering et al.15 showed that infliximab induces apoptosis of peripheral blood (PB) monocytes from healthy volunteers and patients with CD in a dose-dependent manner by activation of caspase-8, 9 and 3 in a Fas-independent manner. ten Hove et al.16 showed that infliximab induces apoptosis of lamina propria T cells in vivo and in the CD3/CD28-activated T-cell line (Jurkat cells). The same group expanded on these observations and showed that in contrast to etanercept, infliximab induces apoptosis of activated PB and lamina propria cells via activation of caspase 3.17 The pro-apoptotic effect of infliximab was also shown by DiSabatino et al.18in vitro on lamina propria T cells and PB T cells from CD patients. This concept of apoptosis induction by infliximab becomes even more attractive in view of data suggesting that a defect in apoptosis is a basic abnormality in the pathogenesis of IBD.19, 20

The aim of this study is to compare the capacity of the newly developed mAb adalimumab to induce apoptosis with that of infliximab and etanercept.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

Reagents and antibodies

Fluorescein isothiocyanate (FITC)-conjugated antihuman CD14 (MφP9, IgG2b), phycoerythrin (PE)-conjugated antihuman CD33 (p67.6, IgG1) and isotype control (X40, IgGγ1), FITC-conjugated annexin V, propidium iodide (Pi) and pan-caspase inhibitor (Z-VAD-FMK) were purchased from BD Biosciences (Erembodegem, Belgium). PE-conjugated mAb Apo2.7 (2.7A6A3) was purchased from Immunotech (Marseille, France). 7-Amino actinomycin-D (7AAD) was obtained from Novabiochem (La Jolla, CA, USA). Human polyclonal immunoglobulins (huIgG; Sandoglobulin) were purchased from Novartis (Basel, Switzerland). Infliximab (Remicade, H98-486, IgG1) was a gift from Schering-Plough (Centocor Inc, Malvern, PA, USA). Adalimumab (Humira M02-497) was a gift from Abbott (Abbott Park, IL, USA). Etanercept (Enbrel) was purchased from Immunex (Seattle, WA, USA). Lipopolysaccharide (LPS) from Escherichia coli, serotype 0111:B4 was obtained from Sigma Chemical Co. (St Louis, MO, USA). Recombinant interferon (IFN)-γ was bought from Roche Diagnostics (Mannheim, Germany).

Isolation of CD33+ monocytes

Heparinized blood was drawn from healthy volunteers or patients with active CD. PB mononuclear cells (PBMCs) were collected by density-gradient centrifugation using Ficoll-Hypaque (Lymphoprep, Nycomed, Oslo, Norway). Monocytes were subsequently obtained by positive selection using anti-CD33-coated MicroBeads (Miltenyi Biotec, Berdisch Gladbach, Germany) and a MACS separator. The procedure resulted in 90% pure CD14+ monocytes as assessed on a FACSort (Becton Dickinson, San Jose, CA, USA).

Detection of antibody binding to monocytes

An indirect immunofluorescence technique was used for detection of adalimumab, infliximab and etanercept binding. PB CD33+ monocytes (1 × 106) were first cultured with LPS (1 μg/mL) at 37 °C/5% CO2 for 6 h, washed three times, then incubated with anti-Fc receptor (FcR)I mAb (10 μg, 197; Medarex, Princeton, NJ, USA) and anti-FcRII mAb (10 μg, 2.4G2; Medarex) at 4 °C for 30 min. Cells were subsequently incubated for 30 min at 4 °C with adalimumab (10 μg), infliximab (10 μg), etanercept (10 μg) or huIgG (10 μg), washed three times, stained with FITC-conjugated goat F(ab’)2-antihuman Ab, and analysed by flow cytometry. A scattergram combining side light scatter and forward light scatter was used to exclude debris and contaminating lymphocytes.

Cell culture and determination of apoptosis by annexin V, Apo2.7 and 7AAD staining

Isolated monocytes were incubated with anti-FcRI mAb (0.1 μg/1 × 106 cells) and anti-FcRII mAb (0.1 μg/1 × 106 cells) at 4 °C for 30 min to block FcR. Cells were washed, adjusted to 0.5 × 106 cells in 1 mL RPMI 1640 medium (Boehringer, Heidelberg, Germany) supplemented with 2 mml-glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% bovine calf serum. A fraction of the monocytes were preincubated with the pan-caspase inhibitor, Z-VAD-FMK (50 μm) for 1 h at 37 °C. Subsequently cells were cultured with LPS 1 μg/mL and respectively with adalimumab (10 μg/mL), infliximab (10 μg/mL), etanecept (10 μg/mL) or huIgG (10 μg/mL). Supernatants were collected after different time periods to measure TNF-α, interleukin (IL)-10 and IL-12 by enzyme-linked immunoabsorbent assay (ELISA). Monocytes were harvested after different times of culture. In a separate experiment, the number of viable monocytes was calculated by trypan blue staining. Cells were washed, resuspended at 5 × 106/mL and incubated with 5 μL annexin V–FITC and Pi, 5 μL Apo2.7–PE-conjugated antibody (Ab) or 5 μL of 7AAD solution (7AAD was dissolved in acetone and diluted in PBS at a concentration of 20 mg/mL) for 10 min at 4 °C protected from light. Samples were immediately analysed with a FacSort without wash or fixation. Data on 10 000 events were acquired and processed using cellquest software.

Measurement of cytokines

The concentrations of IL-10, IL-12 (p70) and TNF-α in culture supernatants were measured with a sandwich ELISA technique using combinations of unlabelled and biotin-coupled MoAb to different epitopes of each cytokine. Coating MoAb for these three assays, respectively, were JES3-9D7 (Pharmingen, San Diego, CA, USA), MAB611 (R&D Systems, Minneapolis, MN, USA) and 68B2B3/68B6A3 (Biosource/Medgenix Diagnostics, Brussels, Belgium). Biotinylated detection mAb were JES3-12G8 (Pharmingen), BAF219 (R&D Systems) and 68B3C5 (Biosource/Medgenix Diagnostics). Human recombinant IL-10, IL-12 and TNF-α were used as standards. The lower detection limit for all cytokines was ≤10 pg/mL.

Statistical analysis

Statistical analysis was performed with prism mac 3.0. All analyses were performed as comparison of experimental groups with respective controls by Mann–Whitney U-testing, or via Kruskall–Wallis testing where appropriate. All tests were two-sided and values of P < 0.05 were considered significant. Data are presented as mean ± S.E.M.

Anti-TNF reagents bind to PB monocytes

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

Results of binding studies of anti-TNF reagents to PB monocytes by immunofluoresence are shown in Table 1. Mean fluorescence intensity (MFI) was significantly higher after either adalimumab, infliximab or etanercept incubation of LPS-activated cells when compared with huIgG-incubated cells (P < 0.05), thus demonstrating binding of these reagents to the cells. LPS stimulation slightly but not significantly enhanced binding of each of the three reagents (Table 1).

Table 1.  Adalimumab, infliximab and etanercept binding to CD33+ PB monocytes mean fluorescence intensitya
 AdalimumabInfliximabEtanercepthuIgG
  1. PB, peripheral blood; LPS, lipopolysaccharide; FcR, Fc receptor; mAb, monoclonal antibody; huIgG, human polyclonal immunoglobulins; FITC, fluorescein isothiocyanate.

  2. a CD33+ PB monocytes were first cultured either in medium alone or with LPS (1 μg/mL) for 6 h. After incubation with blocking anti-FcRI mAb (10 μg) and anti-FcRII mAb (10 μg) (to exclude FcR binding), cells were incubated with either adalimumab (10 μg), infliximab (10 μg), etanercept (10 μg) or huIgG (10 μg) respectively and subsequently stained with FITC-conjugated goat F(ab’)2-antihuman IgG antibody. Cells were analysed by flow cytometry; 10 000 events were acquired and processed using cellquest software. The data shown are mean ± S.E.M. of mean fluorescence intensity from three independent experiments (*P < 0.05, compared with huIgG binding).

Without LPS205 ± 62*167 ± 55179 ± 43*87 ± 43
With LPS283 ± 43*252 ± 78*239 ± 70*121 ± 69

Adalimumab and infliximab induce apoptosis of PB monocytes

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

Induction of apoptosis in vitro was studied with three different methods: annexin V–Pi, Apo 2.7 and 7AAD. These three assays analyse apoptosis at the level of the cell membrane, mitochondria and nucleus respectively.21–23 We used huIgG as a negative control. Figure 1 exemplifies for the three different methods that apoptosis occurred in monocytes from a healthy donor after incubation in vitro with infliximab or adalimumab. The 48 h kinetic study on PB monocytes shown in Figure 2 further illustrates that a large proportion of the monocytes incubated with adalimumab or infliximab were apoptotic, while only 0–5% of the monocytes were apoptotic in etanercept- and huIgG-treated cultures. Enhanced apoptosis was detectable already after 2 h. The time-point at which the highest level of apoptosis could be measured depended on the method used. Annexin V (+) and Pi (−) detected maximal apoptosis between 12 and 30 h, while Apo2.7 detected maximal apoptosis between 2 and 18 h and 7AAD between 6 and 30 h. Viable cell number gradually decreased to approximately 50% after incubation for 48 h with adalimumab or infliximab, and to approximately 85% after incubation with etanercept or huIgG (Table 2).

image

Figure 1. Adalimumab and infliximab induce apoptosis of monocytes. CD33+ monocytes were prepared and cultured for 6 (b, e and h) or 18 (a, c, d, f, g and i) h with lipopolysaccharide (LPS) and either adalimumab, infliximab, etanercept or human polyclonal immunoglobulins (huIgG) as described in Materials and Methods. Apoptosis was analysed by staining with Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (a, d and g), Apo2.7–phycoerythrin (PE)-conjugated antibody (b, e and h), 7-amino actinomycin-D (7AAD) solution (c, f and i). Samples were analysed with a FacSort. Data on 10 000 events were acquired and processed using cellquest software. Numbers shown in each figure from the gated area (M1) represents percentage of [Annexin V (+), Apo2.7 (+) or 7AAD (+)] CD33+ monocytes (experimental situation/control situation). (a, b and c): CD33+ monocytes cultured with adalimumab (filled histograms) or huIgG (open histograms); (d, e and f): CD33+ monocytes cultured with infliximab (filled histograms) or huIgG (open histograms); (g, h and i): CD33+ monocytes cultured with etanercept (filled histograms) or huIgG (open histograms). The data shown are representative for at least six independent experiments.

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image

Figure 2. Kinetic analysis of apoptosis of CD33+ monocytes. CD33+ monocytes were cultured for 48 h and analysed for apoptosis at different time-points as described in the legend of Figure 1. The results represent the mean ± S.E.M. of at least five independent experiments for each condition. In (a), cells were incubated with adalimumab, infliximab or human polyclonal immunoglobulins (huIgG) as indicated; in (b), cells were incubated with etanercept, infliximab or huIgG. Data in (a) and (b) are from different experiments. Significant differences were calculated at each time-point (*P < 0.05, **P < 0.01, ***P < 0.001).

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Table 2.  Survival rate of CD33+ PB monocytes after incubation with adalimumab, infliximab or etanercepta
Incubation periodAdalimumabInfliximabEtanercepthuIgG
  1. PB, peripheral blood; LPS, lipopolysaccharide; FcR, Fc receptor; mAb, monoclonal antibody; huIgG, human polyclonal immunoglobulins.

  2. a CD33+ PB monocytes were first cultured with anti-FcRI mAb (10 μg) and anti-FcRII mAb (10 μg). Subsequently cells were washed, adjusted to 0.5 × 106 cells/mL and incubated with either adalimumab (10 μg/mL), infliximab (10 μg/mL), etanercept (10 μg/mL) or huIgG (10 μg/mL) respectively. LPS (1 μg/mL) was added to all the cultures. After different time period culture, cells were counted and non-viable cells were excluded by trypan blue staining. The data are expressed as percentage of the initial cell number. The mean ± S.E.M. are shown from three independent experiments (*P < 0.05, **P < 0.01, compared with huIgG).

 6 h98 ± 298 ± 299 ± 2101 ± 3
18 h72 ± 6*74 ± 3**92 ± 197 ± 2
36 h55 ± 6**54 ± 7*88 ± 291 ± 5
48 h42 ± 5*45 ± 5**82 ± 485 ± 3

Apoptosis induced by adalimumab and infliximab is caspase-dependent

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

To evaluate whether apoptosis induced by adalimumab and infliximab is caspase-dependent, we introduced the pan-caspase inhibitor, Z-VAD-FMK24, 25 to the cultures. As shown in Figure 3, we found that preincubation of monocytes with Z-VAD-FMK significantly down-regulated apoptosis induction with either adalimumab or infliximab. Moreover, this inhibition of apoptosis induction could be confirmed with each of the three methods.

image

Figure 3. Apoptosis induced by adalimumab and infliximab is caspase-dependent. CD33+ monocytes were prepared and cultured as explained in the legend of Figure 1. Part of them was preincubated with Z-VAD-FMK (50 μm) for 1 h as indicated. Thereafter, adalimumab, infliximab or human polyclonal immunoglobulins (huIgG) were added, and 18 h later apoptosis was analysed by staining with Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide, Apo2.7–phycoerythrin (PE)-conjugated antibody or 7-amino actinomycin-D (7AAD) solution. Lines connect data from the same donor with or without Z-VAD-FMK (*P < 0.05, **P < 0.01, ***P < 0.001). Immunoglobulin (Ig)G, human IgG; Z, Z-VAD-FMK; I, infliximab; A, adalimumab.

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Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

We next analysed the apoptosis inducing effect of adalimumab and infliximab on monocytes from CD patients. We investigated apoptosis of monocytes from patients with active CD at one time-point only: 12 h. Both anti-TNF antibodies induced monocyte apoptosis as detected by either method (data not shown). We could not find significant differences in susceptibility to apoptosis between CD patients and healthy donors. After treatment with etanercept no evidence of apoptosis induction above background was obtained (data not shown).

Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

Finally, we studied the effect of anti-TNF reagents on cytokine production by LPS-stimulated monocytes: IL-10 and IL-12. Although all three reagents efficiently neutralized TNF, we found that IL-10 and IL-12 production were down-regulated only by adalimumab and infliximab (Figure 4). Also in cultures of CD patients’ monocytes, after culture for 12 h, IL-10 production was significantly down-regulated by adalimumab and infliximab but not by etanercept (not shown).

image

Figure 4. Interleukin (IL)-10 and IL-12p70 production by monocytes are down-regulated by adalimumab and infliximab. Peripheral blood (PB) CD33+ monocytes (0.5 × 106) were incubated with anti-Fc receptor (FcR)I monoclonal antibody (mAb) (0.1 μg) and anti-FcRII mAb (0.1 μg), then cultured with adalimumab (10 μg/mL), infliximab (10 μg/mL), etanercept (10 μg/mL) or huIgG (10 μg/mL) as indicated. Lipopolysaccharide (LPS) (1 μg/mL) was added in all culture conditions. Supernatants were collected after different time periods and enzyme-linked immunoabsorbent assay (ELISA) was used to detect cytokines. The results represent the median value of at least five experiments. Significances were calculated at each time-point (*P < 0.05, **P < 0.01, ***P < 0.001).

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We can conclude from this that TNF neutralization by itself does not affect IL-10 and IL-12 production. However, both adalimumab and infliximab inhibit IL-10 and IL-12 production most likely as a result of a direct effect on monocytes.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References

Apoptosis induction potentially plays a major role in the induction of remission of CD by anti-TNF therapy. We here report that adalimumab, a newly developed humanized mAb to TNF, induces apoptosis of monocytes in vitro, to the same extent as infliximab. Both antibodies induce monocyte apoptosis by a caspase-dependent pathway as demonstrated by the use of the pan-caspase inhibitor, Z-VAD-FMK. In contrast, etanercept-treated monocytes, similar to the huIgG-treated cells, survived in vitro and only very low percentages (approximately 5%) of dead cells were detected, probably reflecting spontaneous apoptosis level. We further found that PB monocytes, no matter whether they originated from healthy donors or CD patients, were equally sensitive to apoptosis induction. We used anti-FcRI mAb and anti-FcRII mAb to block these IgG receptors expressed on the monocytes,26 in order to exclude Ab Fc binding to monocytes in the process of apoptosis induction. The possible induction of apoptosis by LPS itself27 at the concentration used for stimulation in our experiments, or by LPS-induced cytokines (IL-10, TNF) was excluded in a preliminary study, in which apoptosis of monocytes cultured with LPS or medium was similar over a 48-h culture period.

Apoptosis can now been dissected at the molecular, biochemical and morphological level using several methods.28 No single method is sufficient to identify apoptotic cell death. In our study, we used annexin V–Pi, Apo2.7 mAb and 7AAD to investigate apoptosis at different cell levels. Annexin V binds to phosphatidylserine of the apoptotic cells21 and the combination with Pi enable us to selectively stain early apoptotic cells. Apo2.7 Ab reacts with a 38-kDa mitochodrial membrane protein in cells undergoing apoptosis22 and 7AAD specifically binds to the broken end of DNA of non-viable cells.23 We proved apoptosis of monocytes by all three markers, but there were differences in sensitivity for early apoptosis detection and in the amount of cells identified as being apoptotic. We found that both annexin V–Pi and Apo2.7 are early markers, but apoptosis could be observed by all three reagents after incubation for 12–18 h. Different markers probably identify cells at different stages of apoptosis. The combination of these methods also makes us confident that apoptosis was indeed the underlying mechanism of cell death, and the use of the pan-caspase inhibitor, Z-VAD-FMK, further corroborates this conclusion.

Etanercept also binds to tmTNF expressing monocytes, but this reagent is monomeric and cannot cross-link tmTNF.29 Scallon et al.29 performed binding studies and proved that infliximab binds to the tmTNF with higher avidity than etanercept and moreover, infliximab formed more stable complexes with tmTNF expressed on transfected cells relative to analogous complexes formed with etanercept. Thus, high-affinity binding and cross-linking of tmTNF is probably required to signal the apoptosis cascade.

We finally also found that LPS-induced IL-10 and IL-12 productions were affected by adalimumab and infliximab. As TNF neutralization with etanercept did not affect IL-10 and IL-12 production, we hypothesize that this down-regulation of IL-10 and IL-12 is due to a direct functional effect of adalimumab/infliximab on the cells, or to a lower amount of living cells after incubation with adalimumab/infliximab.

In the human inflamed gut both macrophages15 and activated T cells17 express high amounts of tmTNF. Functional modulation and/or killing of those cells probably contribute to the beneficial effect of anti-TNF antibodies. We still do not know which cell type is the main target of anti-TNF therapy, and studies should further correlate clinical effectivity of anti-TNF reagents to their apoptosis-inducing capacity.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Anti-TNF reagents bind to PB monocytes
  7. Adalimumab and infliximab induce apoptosis of PB monocytes
  8. Apoptosis induced by adalimumab and infliximab is caspase-dependent
  9. Adalimumab and infliximab induces apoptosis of PB monocytes from CD patients
  10. Adalimumab and infliximab down-regulate IL-10 and IL-12p70 production of monocytes
  11. Discussion
  12. Acknowledgements
  13. References
  • 1
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  • 2
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    Targan SR, Hanauer SB, van Deventer SJ, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med 1997; 337: 102935.
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    Stack WA, Mann SD, Roy AJ, et al. Randomised controlled trial of CDP571 antibody to tumour necrosis factor-alpha in Crohn's disease. Lancet 1997; 349: 5214.
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    Sandborn WJ, Feagan BG, Hanauer SB, et al. An engineered human antibody to TNF (CDP571) for active Crohn's disease: a randomized double-blind placebo-controlled trial. Gastroenterology 2001; 120: 13308.
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    Feagan BG, Sandborn WJ, Baker JP, et al. A randomized, double-blind, placebo-controlled, multi-center trial of the engineered human antibody to TNF (CDP571) for steroid sparing and maintenance of remission in patients with steroid-dependent Crohn's disease. Gastroenterology 2000; 118: A655 (Abstract).
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    Evans RC, Clarke L, Heath P, et al. Treatment of ulcerative colitis with an engineered human anti-TNF-alpha antibody CDP571. Aliment Pharmacol Ther 1997; 11: 10315.
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    Schreiber S, Rutgeerts P, Fedorak R, et al. CDP870, a Humanised anti-TNF Antibody Fragment, Induces Clinical Response with Remission in Patients with Active Crohn's Disease (CD). Orlando, FL, USA: Digestive Disease Week, 2003: 102090 (Abstract).
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    Sandborn WJ, Hanauer SB, Katz S, et al. Etanercept for active Crohn's disease: a randomized, double-blind, placebo-controlled trial. Gastroenterology 2001; 121: 108894.
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