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. 2008 Dec;154(3):325–331. doi: 10.1111/j.1365-2249.2008.03773.x

Non-response to infliximab may be due to innate neutralizing anti-tumour necrosis factor-α antibodies

E C Ebert *, K M Das *, V Mehta ‡, C Rezac †
PMCID: PMC2633230  PMID: 18826498

Abstract

Infliximab is a chimeric anti-tumour necrosis factor (TNF)-α antibody that is therapeutic in many patients with inflammatory bowel disease. What causes certain patients not to respond is unknown. The question posed is whether innate anti-TNF-α antibodies play any role in the response to infliximab. Blood was drawn prior to the initial dose of infliximab. Serum anti-TNF-α antibodies were quantitated by enzyme-linked immunosorbent assay (ELISA). Affinity-purified anti-TNF-α antibodies were isolated from serum immunoglobulin G using TNF-α-coated beads. The ability of these antibodies to induce apoptosis of macrophages was measured by annexin and propidium iodide staining. Changes in TNF receptor type 2 (TNFR2) expression and release were determined by immunofluorescence and ELISA respectively. TNF-α-neutralization was assessed by the reversal of the lytic actions of TNF-α on WEHI cells. The amounts of innate anti-TNF-α antibodies in the serum from infliximab responders versus non-responders were the same. Apoptosis of monocytes increased with infliximab and by several of the purified anti-TNF-α antibodies, but these findings did not vary with the patients' responses to infliximab. Effects of the anti-TNF-α antibodies on the expression of TNFR2 on monocytes and their release of soluble TNFR2 did not vary with the patients' responses to infliximab. However, the neutralizing capacity of these antibodies differed, with responders having antibodies that reduced only 47 ± 4% of the TNF-α activity while those from non-responders reduced 70 ± 5% of the TNF-α activity (P < 0·01). Non-responders have innate anti-TNF-α antibodies with greater neutralizing activity than antibodies from responders. Any TNF-α-mediated disease process would be neutralized by intrinsic antibodies, so that the disease is likely to be driven by non-TNF-α-mediated events.

Keywords: Crohn's disease, inflammatory bowel disease, tumour necrosis factor, ulcerative colitis

Introduction

Tumour necrosis factor (TNF)-α is a cytokine produced by many cell types, including T cells and macrophages, cells whose numbers are increased in the intestinal mucosa of patients with Crohn's disease (CD). TNF-α is essential in the defence against mucosal pathogens [1] but also contributes to destructive inflammation. An animal model with dysregulated TNF-α release results in chronic inflammatory arthritis and a Crohn's-like inflammatory bowel disease (IBD) [2].

Two distinct but related receptor molecules, type I (p60) and type II (P80), share less than 25% overall amino acid sequence identity, with most of the homology found in the extracellular domain [3]. Their cytoplasmic domains differ, indicating separate intracellular signaling pathways. These receptors are recruited to the surface with stimulation and are either shed or internalized [4]. Soluble receptors may inactivate TNF-α or serve as a sink, continually providing cytokine as levels decrease. Type 1 TNF receptor (TNFR1) mediates apoptosis initiated by membrane TNF-α on the effector cell. Type II receptor (TNFR2), produced in larger amounts than TNFR1, may serve to capture TNF-α for processing by TNFR1 [5]. TNFR2 or structurally similar receptors are promoted by mycobacteria in order to promote their survival [6]. This suggests that TNFR2 serves an important role in vivo.

The pathogenesis of CD in a large subset of patients involves TNF-α as evidenced by the therapeutic effects of infliximab, a chimeric monoclonal antibody against TNF-α. Infliximab has several possible mechanisms of action. It immediately neutralizes TNF-α. In addition, it induces apoptosis of macrophages and lamina propria lymphocytes (LPLs) in the colon of patients with CD [7,8]. Such an effect is due probably to the binding of the drug to TNF-α on the cell surface, resulting in outside-to-inside reverse signalling [9]. Furthermore, infliximab decreases secretion of cytokines, including interleukin (IL)-1, IL-6, IL-8 and interferon (IFN)-γ[10–19]. Soluble TNFR2 levels are increased in patients with active CD and reduced with infliximab therapy [12,17].

Naturally occurring antibodies against cytokines have been identified in certain autoimmune diseases (such as rheumatoid arthritis and systemic lupus erythematosus) and in infectious diseases (such as hepatitis and varicella) [18,19]. They are sometimes found in normal individuals. Human immunoglobulin (Ig), used to treat immunoinflammatory diseases, contains IgG directed against IL-1α, IL-6, IFN-α and granulocyte–macrophage colony-stimulating factor [20,21], while the presence of IgG against TNF-α is controversial [20,22]. Anti-cytokine antibodies have not been described in IBD.

Whether anti-cytokine antibodies are epiphenomena or serve a function has not been determined. They may arise because of an imperfect discrimination between self and non-self that controls the balance between immunity and autoimmunity [18,19]. Alternatively, there may be a break in tolerance by molecular mimicry because of such molecules as the IL-10-like protein encoded by some poxviruses [18]. Anti-cytokine antibodies may act as carrier proteins for cytokines, extending their circulation time from minutes to hours. This would be due to protection against proteolytic degradation as well as delayed glomerular filtration by the kidney. In addition, these antibodies may buffer rapid changes in the concentrations of cytokines. Finally, some, but not all, anti-cytokine antibodies have neutralizing activity [20].

The present report quantitates the amounts and analyses the functions of innate anti-TNF-α antibodies from patients with IBD. The questions posed are whether they mimic or interfere with the actions of infliximab and whether they are involved in responsiveness to infliximab.

Methods

Patient populations

For measurement of anti-TNF-α levels by enzyme-linked immunosorbent assay (ELISA), serum samples were obtained after informed consent from normal individuals and from patients with CD and ulcerative colitits (UC), both active and in remission, who were not on infliximab. Immunosuppressant therapy was defined as the use of 6-mercaptopurine, azathioprine or prednisone (greater than 15 mg daily).

For isolation of anti-TNF-α antibodies, serum samples were collected from patients with active CD [CD Activity Index (CDAI) of greater than 180] or active UC [Disease Activity Index (DAI) of greater than 3] just before their first exposure to infliximab treatment. The patients were followed prospectively, and their clinical response after the usual course of infliximab assessed 8 weeks later. Those with less than a 75-point drop in their CDAI or less than a 2-point drop in their DAI were considered to be non-responders.

Anti-TNF-α ELISA

Flat-bottomed microtitre wells were coated overnight at 4°C with TNF-α (R&D Systems, Minn, MN, USA) suspended in 50 mM sodium carbonate buffer (pH 9·6). The excess cytokine was removed by washing with phosphate-buffered saline (PBS). RPMI-1640 with 10% fetal calf serum (0·04 ml) (Gibco, Grand Island, NY, USA) was then added to each well for blocking. Test serum samples (1:200 dilution final concentration) were added in duplicate, and the plate was incubated for 1 h at room temperature before washing with PBS. This dilution provided the greatest difference between IBD and Nl serum values. The next step was the addition of goat anti-human IgG or IgM conjugated to alkaline phosphatase (1:20 000 dilution), followed by a 1-h incubation. After washing with PBS, phosphatase substrate solution (Sigma-Aldrich, St Louis, MO, USA) was introduced and, after 30 min, colour development [optical density (OD)] was read spectrophotometrically at absorbance of 405 nm. The control value (without patient serum) was subtracted from each test value to obtain the final reading. Each serum sample was tested in at least three different experiments and the results averaged. In general, the intra-experimental variation in OD levels was 0·015 ± 0·005, while the interexperimental variation was 0·025 ± 0·01.

Isolation of anti-TNF-α and anti-IL-2 antibodies

The IgG fractions were isolated from serum using a Protein G column. Magnetic beads (Dynal Biotech ASA, Oslo, Norway) were coated with TNF-α or IL-2 following the manufacturer's directions. Then, the IgG fractions were incubated with the cytokine-coated beads. The beads were washed thoroughly, then exposed to a 0·1 M glycine HCl buffer (pH 3) for 5 min. The eluted cytokine-specific IgG was saved, neutralized, concentrated and protein content determined.

Apoptotic effects of anti-TNF-α antibodies

To obtain monocytes, peripheral blood lymphocytes (PBLs) were isolated by Ficoll density gradient centrifugation. Then, the PBLs (10 × 106/ml) were incubated in wells and the non-adherent cells removed. The adherent monocytes were incubated with phorbol myristate acetate (PMA, 50 ng/ml) in the presence or absence of infliximab (Centocor, Inc., Horsham, PA, USA), anti-TNF-α and/or anti-IL-2 antibodies (all at 20 µg/ml). After 18 h, the cells were recovered by trypsin–ethylenediamine tetraacetic acid treatment, then stained with annexin conjugated to fluorescein isothiocyanate with propidium iodide (PI). The resulting immunofluorescence (IF) was read by flow cytometry (Beckman Coulter FC500, Miami, FL, USA).

Measurement of TNFR2 expression and release

Monocytes were incubated for 18 h in the presence or absence of infliximab, anti-TNF-α and/or anti-IL-2 antibodies (20 µg/ml). They were then stained by IF for TNFR2 (antibody from R&D Systems) and fluorescence read by flow cytometry. The culture medium was tested for soluble TNFR2 by ELISA (R&D Systems).

Neutralizing effects of anti-TNF-α antibodies as determined by the mitochondrial activity measured in the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay

WEHI cells (ATCC, Manassas, VA, USA), which are highly susceptible to lysis by TNF-α, were incubated for 18 h in microtitre plates with TNF-α alone (10 ng/ml; R&D Systems) or with TNF-α pre-exposed to infliximab, anti-TNF-α, and/or anti-IL-2 antibodies (20 µg/ml). The numbers of viable cells were quantitated by the MTT (Sigma-Aldrich) assay. That is, MTT (0·2 mg/ml) was added to each microwell, and the cells incubated for 6 h at 37°C. Colour development was initiated by the addition of dimethyl sulphoxide (0·1 ml). The amount of yellow MTT that was reduced to purple formazan was measured spectrophotometrically at an absorbance of 550 nm. This reduction takes place only when the mitochondrial dehydrogenase enzyme cleaves the tetrazolium rings, a process that is related directly to the numbers of viable cells.

Statistical analysis

Two sets of data were compared using the Student's t-test (unpaired variables).

Results

The amounts of anti-TNF-α IgG antibodies in the serum of infliximab-naive UC and CD patients and normal individuals were quantitated by ELISA (Fig. 1). The average levels were the same for the three groups. However, there were fractions of patients (12% of UC patients and 22% of CD patients) with levels greater than those of all normal individuals. The levels did not correlate with disease activity, disease duration or the use of immunosuppressive medications. Anti-TNF-α IgM antibodies, in contrast, were nearly undetectable for all three patient groups.

Fig. 1.

Fig. 1

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Anti-tumour necrosis factor (TNF)-α immunoglobulin (Ig)G and IgM antibodies were measured in the serum of patients with ulcerative colitis (UC) or Crohn's disease (CD) or from normal individuals (NI) by enzyme-linked immunosorbent assay. The dotted line indicates the highest value reached by all NI.

To determine whether the amounts of anti-TNF-α antibodies differed for responders versus non-responders to infliximab, levels of these antibodies were measured by ELISA using serum samples from patients just before their first dose of infliximab and the results correlated with response to the drug determined 8 weeks later (Fig. 2). The responders and non-responders were divided equally between UC and CD. There were no differences in the average levels of anti-TNF-α antibodies between the responders and non-responders to infliximab or between CD and UC. Again, a fraction of patients (23% of responders and 23% of non-responders) had levels greater than those of all normal individuals. The few values in Fig. 1 greater than the OD reading of 0·2 corresponded to patients who had not received infliximab, so they are absent in Fig. 2.

Fig. 2.

Fig. 2

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Tumour necrosis factor (TNF)-α immunoglobulin (Ig)G antibodies were measured in the serum of responders and non-responders to infliximab. The dotted line indicates the highest value reached by all normal individuals (NI) in Fig. 1. Squares with lines indicate those individuals from whom anti-TNF-α antibodies could not be purified, both ulcerative colitis (UC) and Crohn's disease (CD). The open squares represent patients with UC, while the solid squares indicate patients with CD.

Because response to infliximab did not correlate with the amounts of anti-TNF-α antibodies, it may correlate instead with their function. For the remainder of the experiments, intrinsic anti-TNF-α or anti-IL-2 antibodies were used from UC and CD patients isolated from serum drawn just before their first dose of infliximab. The black data points in Fig. 2 indicate the patients from whom anti-TNF-α antibodies could not be isolated as the levels were low. There was an association between the amounts of anti-TNF-α antibodies or infliximab compared with the OD reading on ELISA (Fig. 3) where the secondary reagent was goat anti-human or goat anti-mouse, respectively, conjugated to alkaline phosphatase.

Fig. 3.

Fig. 3

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Anti-tumour necrosis factor (TNF)-α antibodies from two patients (results averaged) and infliximab were measured by enzyme-linked immunosorbent assay for their corresponding optical density values. The results (mean ± standard error of the mean) were no different between the two.

The main mechanism of action by infliximab is thought to be the initiation of apoptosis. Therefore, monocytes, stimulated with PMA, were co-cultured with infliximab and/or with purified anti-TNF-α or anti-IL-2 antibodies (20 µg/ml each) isolated from the IBD patients and the percentage of apoptosis and/or necrosis determined by annexin and PI staining (Figs 4 and 5). The percentages of annexin+ and/or PI+ cells were the same for the responders versus non-responders and for UC versus CD patients. The average percentages were greater than those induced by medium alone. This indicates that the anti-TNF-α antibodies induce apoptosis but that it did not correlate with response to infliximab. When anti-TNF-α antibodies from three responders and three non-responders were combined individually with infliximab, the percentages of annexin+ PI+ cells were similar to the highest value induced either by the antibodies or by infliximab alone (not shown) This indicates that the anti-TNF-α antibodies neither synergize nor block the effects of infliximab. Intrinsic anti-IL-2 antibodies from three patients with CD induced results similar to medium alone, suggesting that the effects of anti-TNF-α antibodies are specific (not shown).

Fig. 4.

Fig. 4

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Annexin and propidium iodide staining of macrophages cultured with anti-tumour necrosis factor-α antibodies from responders (top three graphs) and non-responders (lower three graphs) are shown. The numbers indicate the percentage of events in each quadrant.

Fig. 5.

Fig. 5

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The ability of purified anti-tumour necrosis factor-α antibodies to induce apoptosis and/or necrosis of phorbol myristate acetate-stimulated monocytes was measured by annexin and propidium iodide staining. The degree of apoptosis/necrosis induced by infliximab or medium alone is indicated on the right. The open squares represent patients with ulcerative colitis, while the solid squares indicate patients with Crohn's disease.

Infliximab and the purified anti-TNF-α antibodies may change TNFR2 expression by monocytes, thereby affecting their response to TNF-α. To evaluate this, monocytes were cultured with or without anti-TNF-α or anti-IL-2 antibodies and/or infliximab, and TNFR2 expression determined 18 h later (Fig. 6). PMA was not used as its effects may mask a similar effect induced by the anti-TNF-α antibodies. Expression was lower with infliximab than with medium alone, suggesting that infliximab reduces TNF-α responsiveness of monocytes by reducing TNFR2 expression. There were no differences with the antibodies compared with that seen with infliximab or medium alone. The combination of anti-TNF-α antibodies and infliximab resulted in expression that was similar to the average of that induced by either one alone. There were no differences between responders and non-responders or between UC and CD. Anti-IL-2 antibodies from three individuals with CD did not affect TNFR2 expression (not shown).

Fig. 6.

Fig. 6

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The ability of purified anti-tumour necrosis factor (TNF)-α antibodies to alter TNF receptor type 2 (TNFR2) expression by monocytes was determined by immunofluorescence. TNFR2 expression induced by infliximab or medium alone is indicated on the right. The open squares represent patients with ulcerative colitis, while the solid squares indicate patients with Crohn's disease.

Perhaps infliximab and the anti-TNF-α antibodies influence the release of soluble TNFR2, an important TNF-α neutralizer in vivo. To evaluate this, monocytes were cultured in the presence of infliximab and/or the anti-TNF-α or anti-IL-2 antibodies, and the supernates collected and tested for soluble TNFR2 by ELISA (Fig. 7). Infliximab tripled the amounts of soluble TNFR2, an action that would promote TNF-α neutralization by this drug. The anti-TNF-α antibodies also increased the amounts. The average levels were the same for the responders and the non-responders, however. Combining anti-TNF-α antibodies and infliximab resulted in a TNFR2 level similar to the highest value reached by the antibodies or infliximab alone. Anti-IL-2 antibodies from three individuals with CD did not affect soluble TNFR2 levels (not shown).

Fig. 7.

Fig. 7

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The ability of purified anti-tumour necrosis factor (TNF)-α antibodies to induce the release of soluble TNF receptor type 2 (TNFR2) was determined by by enzyme-linked immunosorbent assay. The amounts of TNFR2 released with infliximab or medium alone are shown on the right. The open squares represent patients with ulcerative colitis, while the solid squares indicate patients with Crohn's disease.

So far, the anti-TNF-α antibodies had the same actions as did infliximab. There were no differences between responder and non-responders or between UC and CD. In addition, the antibodies did not alter the action of infliximab, indicating that they are not blocking antibodies. The final test was the ability of infliximab and the anti-TNF-α antibodies to neutralize TNF-α as measured using a cell line that is lysed by TNF-α. To evaluate this, TNF-α was preincubated with the infliximab and/or anti-TNF-α or anti-IL-2 antibodies and the combination added to the WEHI cells; after 18 h, the numbers of viable cells were determined using the MTT assay (Fig. 8). Cell viability declined by 31 ± 10% with TNF-α. Antibodies from responders had significantly decreased (P < 0·01) neutralizing activity compared with those from non-responders. The non-responders had the same degree of neutralization as infliximab. Again, there was no difference between UC and CD, nor did anti-IL-2 antibodies from three individuals with CD neutralize TNF-α (not shown). Combining anti-TNF-α antibodies withinfliximab had similar neutralizing ability to the average of the two tested alone. Neutralizing TNF-α may be an important action of infliximab whose effects are reduced in the presence of innate neutralizing TNF-α antibodies. The neutralizing capacity of these innate antibodies did not correlate with their amounts in the serum, indicating that they truly differ in function (Fig. 9).

Fig. 8.

Fig. 8

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The ability of purified anti-tumour necrosis factor (TNF)-α antibodies to neutralize the lytic action of TNF-α toward the WEHI cells was measured by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. The neutralizing effects of infliximab or medium alone are shown on the right. The open squares represent patients ulcerative colitis, while the solid squares indicate patients with Crohn's disease.

Fig. 9.

Fig. 9

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Lack of correlation between amounts of anti-tumour necrosis factor (TNF)-α antibodies and TNF-α neutralizing activity. The open squares represent patients with ulcerative colitis, while the solid squares indicate patients with Crohn's disease.

Discussion

The IBD is likely to be due to a variety of immune abnormalities, some of which are ameliorated by infliximab. This study shows that non-responders have anti-TNF-α antibodies with greater neutralizing capacity than those from responders. Although both have active disease, the non-responders have a disease process that is not reversed by TNF-α neutralization provided by infliximab. Any TNF-α-sensitive disease process that they may have would already be neutralized by intrinsic antibodies. No differences were uncovered between UC and CD patients. Anti-IL-2 antibodies did not alter any of the functions tested indicating specificity. Anti-TNF-α antibodies did not alter the effects of infliximab, indicating that they are not blocking antibodies.

Infliximab induces apoptosis of activated T cells and LPLs from patients with CD [7,8]. It has been surmised that infliximab binds membrane (m)TNF-α causing reverse signalling. Using a mTNF-α-transfected Jurkat cell line, infliximab induces more apoptosis than does etanercept [9]. In these experiments, soluble TNF-α was thought not to be involved, as only low levels could be measured using cells lacking TNF receptors. Because infliximab forms a more stable complex with mTNF on transfected cells with higher avidity than does etanercept [23,24], infliximab might transmit stronger signals through mTNF. These Jurkat cells express more mTNF-α than do LPLs, although the authors state that those cells with lower expression also underwent apoptosis to a greater extent with infliximab than with etanercept. It is difficult in this model to know whether this mimics the in vivo situation and whether the same results would be obtained with monocytes. In addition, it does not eliminate the possibility that infliximab action also depends upon its TNF-α neutralizing action.

It has been suggested that infliximab's main action is not TNF-α neutralization as etanercept, with equivalent neutralizing ability, is ineffective in CD. However, there are several reasons why etanercept may be ineffective. First, it is possible that higher doses of this drug would be efficacious. Secondly, the clearance of etanercept is about 13 times higher than for infliximab [25]. Furthermore, subcutaneous administration of etanercept results in lower peak levels than intravenously administered infliximab.

The present study shows that infliximab and the anti-TNF-α antibodies increase in the release of soluble TNFR2. Previous studies have shown that infliximab reduces the serum levels of soluble TNFR2 [12,17]. This could be due to a decrease in the numbers of monocytes as the inflammation subsides. (Monocytes express and secrete much more TNFR2 than do lymphocytes.)

The only function of the anti-TNF-α antibodies that correlated with response was their neutralization of TNF-α-induced lysis of WEHI cells. This is the first time that neutralizing anti-TNF-α antibodies have been described and the first time that anti-cytokine antibodies have been investigated in IBD. The fact that non-responders had anti-TNF-α antibodies with strong neutralizing activity suggests that, in certain individuals, this role of infliximab may be relatively ineffective. Measurement of anti-TNF-α neutralizing antibodies, however, is too cumbersome to be translated into a practical means of distinguishing between responders and non-responders.

In the presence of neutralizing anti-TNF-α antibodies, one would expect the TNF-α levels to be predictive of a clinical response to infliximab. However, TNF-α levels have not been consistently predictive [26–28], probably because they depend upon several factors, including numbers of TNF-α-producing cells and the intrinsic anti-TNF-α antibodies. It is unknown how much the TNF-α activity is neutralized by innate anti-TNF-α antibodies in vivo and whether infliximab responses are curtailed by such antibodies. There are likely to be many reasons for infliximab non-responsiveness, with innate neutralizing anti-TNF-α antibodies being one of the possibilities.

Acknowledgments

This study was supported in part by an Investigator-Sponsored Study Program by AstraZeneca and by the NIH.

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