Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2016 May 12;185(1):72–80. doi: 10.1111/cei.12788

Label‐free detection of immune complexes with myeloid cells

Z Szittner 1,2,✉, A E H Bentlage 3, P Rovero 4,5, P Migliorini 4,6, V Lóránd 7, J Prechl 2, G Vidarsson 3
PMCID: PMC4908290  PMID: 26953930

Summary

The aim of this study was to provide proof‐of‐concept for quantitative and qualitative label‐free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA‐specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme‐linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen‐specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen‐specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2‐based serological positivity and European League Against Rheumatism (EULAR) criteria‐based clinical RA diagnosis. Immunoglobulin‐triggered binding of monocytoid cells can be monitored using a label‐free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti‐citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc‐receptor‐expressing cells is also affected by post‐translational modification of the immunoglobulins.

Keywords: ACPA, Fc receptor, IgG, imaging SPR, monocyte

Introduction

Detection of antigen‐specific antibody reactivity is of great interest for the diagnosis of various pathogenic conditions. Most commonly this is obtained by measuring antibodies in biological samples, such as from blood serum by enzyme‐linked immunosorbent assay (ELISA) 1 or immunofluorescence‐based tests 2. The results obtained represent a simplified view of the immunological reactivity, as only the isotype and/or immunoglobulin G (IgG) subclass of the antigen‐specific antibody is reported as a biomarker of a given pathological state.

However, the diversity of antigen‐specific reactivity is much more complex, as the magnitude of the effector functions is dependent upon the combination of isotypes and subclasses involved and the antibody levels mounted. The antibody glycosylation in the conserved N‐linked glycosylation site at position 297 in the Fc portion in IgG and IgE can be varied, and affects binding affinities to Fc‐receptors 3, 4, 5. IgG and IgM responses can also lead to activation of the complement system 6, and further opsonization of innate molecules such as pentraxins 7 can influence the outcome of the immune response. The resulting immune complex (IC) will lead eventually to differential engagement of Fc‐ and complement receptors on myeloid effector cells. Thus, the final makeup of IC determines the type and magnitude of the resulting inflammatory response 8.

In our previous work we have shown that ICs can be detected through the binding of fluorescently labelled U937 histiocytic lymphoma cells 9 to spotted immunoglobulins and antigens (after serum treatment) on nitrocellulose‐based microarrays 10. U937 cells express the IgA receptor (FcαR), but also the IgG receptors (FcγR) FcγRI and FcγRIIa (CD64 and CD32, respectively) 11. Their different affinities towards the human IgG subclasses 12 resulted in distinct adhesion profiles depending on the IgG subclass, which was also affected by complement deposited by these IgG antibodies.

In the current study we report a novel approach where immunological reactivity of serum samples is detected directly and in real time by the adhesion of immunologically relevant effector cells to immune complexes in a label‐free manner using surface plasmon resonance (SPR). Besides the typical measurements such as determination of affinity, specificity and binding kinetics, we and others have shown that cells can also be detected 13, 14, 15, 16, 17. This has been achieved by coupling cellular ligands (e.g. cell surface antigen‐specific antibodies) covalently onto the sensor and cells are used as analytes, as presented for T and B lymphocytes 14 and breast cancer cell lines 17, or by monitoring morphological changes in real time upon activation 18, 19.

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by autoantibody production 20. These autoantibodies are categorized into two main groups: rheumatoid factors and anti‐citrullinated protein antibodies (ACPA). Rheumatoid factors (RF) are usually IgM antibodies directed against the constant region of IgG. Tests determining IgM RF levels have a relatively low specificity (85%), but are used routinely for their sensitivity (69%) 21. In a recent prospective study, RF positivity was found to be correlated positively with the subsequent onset of RA 22 and was reported to have a higher affinity to hypogalactosylated IgG 23. Moreover, ACPA were also found to be hypogalactosylated compared to total IgG1 24, 25. ACPA positivity of multiple isotypes precedes the onset of the clinical symptoms in RA 24, and the presence of such autoantibodies is now included in the classification criteria of RA 26. ACPA testing is performed routinely with cyclic citrullinated peptide (CCP) ELISA 27. With this technique, isotype distribution can also be monitored; however, no correlations have been found with disease activity. Importantly, ACPA‐triggered, FcγR‐mediated inflammatory cytokine production by macrophages has been demonstrated, indicating a pathogenic role for these antibodies 28. Detection of ACPA by SPR imaging has also been established previously, demonstrating that biomolecular interactions between citrullinated peptides and autoantibodies can be monitored with great precision in real time using this technique 29.

In this study we tested whether the ICs formed on sensor surface with RA‐specific antigens trigger adhesion of U937 cells. We then tested if this quantitative information on both specific antibodies and their capacity to trigger cellular activation obtained simultaneously in a label‐free manner yields useful information regarding the patient's clinical state.

Materials and methods

Cell culture

Human premonocytic cell line U937 was cultured in RPMI‐1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml), and was maintained at 37°C in a humidified atmosphere of 5% carbon dioxide.

Measurement of U937 binding to IgG subclasses and on‐sensor formed complexes with IBIS MX96

The IBIS MX96 (Ibis Technologies, Enschede, the Netherlands) surface plasmon resonance imager (SPRi) has several advantages, as an array with up to 96, but more conveniently 48, different molecules can be coupled to the sensor surface, monitoring cellular interaction to all of them in parallel in real time. The microfluidic system of IBIS MX96 with the sensor surface on the bottom facing upwards in the optical imaging chamber enables the application and gravitational sedimentation of cells, as demonstrated for red blood cell typing and detection of cell antigens on adherent cells 16, 17. Human IgG1–4 (Sigma, St Louis, MO, USA) were spotted in 10 mM acetate buffer, pH 5·4 in replicates to preactivated Easy2Spot P‐type chips (Ssens, Enschede, the Netherlands) using a continuous flow microspotter (Wasatch Microfluidics, Salt Lake City, UT, USA). One hundred nM bovine serum albumin (BSA) in 10 mM sodium acetate buffer, pH 4·0 was spotted as negative control and acetate buffer as reference. Multiple antigenic peptides (MAPs), histone citrullinated peptide 2 (HCP2) 30 and Epstein–Barr virus‐derived viral citrullinated peptide 2 (VCP2) 31 (from Toscana Biomarkers Srl, Siena, Italy), were spotted at 10 mM and 1 mM spots in replicates in 10 mM acetate buffer, pH 5·0 and 10 mM MES buffer, pH 6·0, respectively, with IgG1 and IgG3 as positive controls. The sensor was blocked with 1 mg/ml BSA in 10 mM sodium acetate buffer, pH 4·0 with 0·075% Tween‐80 and then with 100 mM ethanolamine, pH 8·0 with 0·075% Tween‐80. Injection of 50 nM anti‐kappa chain‐specific antibody (mouse anti‐human kappa UNLB, clone SB81a; Southern Biotech, Birmingham, AL, USA) was used to determine equal spotting concentration of the human IgG subclasses, identified as 108 nM for IgG1, 125 nM for IgG2, 100 nM for IgG3 and 148 nM for IgG4. As system and sample buffer, phosphate‐buffered saline (PBS) with 0·1% BSA (PBS–BSA) was used. All materials, including cells and serum samples, were diluted in this buffer. For regeneration between measurements, 200 nM H3PO4 and 0·5% sodium dodecyl sulphate (SDS) was used in two cycles to regenerate the sensor surface. The temperature was set to 25°C. Each chip was not used for more than 14 runs.

For testing serum followed by U937‐myeloid cell reactivity to the spotted antigenic peptides, the baseline (0–300 s) was set with system buffer; then 100 μl 1 : 10 diluted heat‐inactivated serum was injected and the sample association took place for 15 min under back‐and‐forth flow. This was followed by the dissociation phase with system buffer under back‐and‐forth flow for 10 min. Next, U937 cells (200 μl of 2 × 106/ml in PBS–BSA) were injected onto the sensor surface and were incubated for 15 min after stopping the flow, allowing cells to sediment and bind specific ligands. Changes in angular‐shift were recorded to all spots in real time, monitoring both reactivity of sera and cells in corresponding phases of the measurement.

Serum samples

Serum samples from RA patients (n = 18) and control serum samples (n = 5) were obtained by venipuncture at the University of Pécs and the University of Pisa and were stored at −70°C until use. The national ethics committees of Hungary and Italy gave their approval to conduct the study with the following contract numbers, respectively: 24973‐1/2012/EKU (658/PI/2012) and 45066/2012. RA patients fulfilled the international rheumatoid arthritis classification criteria 26. Serum samples were tested with anti‐cyclic citrullinated peptide 2 (CCP2) ELISA (Inova Diagnostics, San Diego, CA, USA), according to the manufacturer's instructions. Heat‐inactivation was carried out at 56°C for 30 min.

Detection of HCP2‐ and VCP2‐specific IgG subclasses in RA samples with ELISA

Ninety‐six‐well ELISA plates were coated overnight at 4°C with 5 µg/ml HCP2 and VCP2, diluted in carbonate–bicarbonate buffer pH 9·6 or PBS, respectively. After washing with PBS + 0·05% Tween‐20, wells were blocked with PBS + 3% BSA for 1 h room temperature (RT), samples were diluted 1 : 50 in PBS with 1% BSA and 0·05% Tween‐20 and were incubated for 3 h on a shaker at RT. Subclass‐specific biotinylated mouse anti‐human antibodies [ahIgG1‐b and 2b (BD Pharmingen, San Diego, CA, USA), ahIgG3‐b and 4‐b (Southern Biotech)] were diluted 1 : 8000 in PBS‐Tween and were incubated for 1 h at RT. Biotinylated antibodies were detected with streptavidin–horseradish peroxidase (HRP) (Sigma), diluted 1 : 5000 in PBS‐Tween for 1 h at RT. After washing, 1% 3,3′,5,5′‐tetramethylbenzidine (TMB) with 0·2% H2O2 in TMB buffer was added to each well. After 10 min, 2N H2SO4 was added to each well to stop the reaction. Optical density was recorded at 450 nm. Control measurements without serum samples were used to determine the background values and background subtracted values were analysed.

Statistical analysis

Spearman's rank correlations were calculated to compare IgG, IgA, IgM and IgG1‐4 and U937 and serum association signals on MAPs. Statistical differences in serum binding and cell adhesion during and following incubation of IgG subclasses and MAPs with healthy and RA sera were assessed by pairwise comparisons of relevant groups using the Mann–Whitney U‐test. To compare the results obtained by cell adhesion to MAPs following serum treatment and results of CCP2 tests and RA diagnosis by a physician, Fisher's exact test was used. Statistical tests were considered significant as follows: non‐significant (n.s.), P ≥ 0·05, *P < 0·05, **P < 0·01 and ***P < 0·001.

Results

U937 cells bind to human IgG1, IgG3 and IgG4 but not to IgG2

To measure serum responses to antigens and cells simultaneously, we utilized a SPR biosensor system allowing monitoring of up to 48 interactions simultaneously on an array of covalently coupled ligands. While serum reaction can be detected easily by classical SPR, we and others have recently shown the IBIS MX96 system to be capable of red blood cells, platelets and tumour cell detection after gravity sedimentation 16, 17, 32 – but never to deposited ligands. To investigate if U937 cells bind IgG in the IBIS, first we determined the coupling efficiency of each IgG subclass, using antibody against the light chain (anti‐kappa monoclonal antibody) shared by all four IgG subclasses. At 100 response units (RUs) of each coupled IgG subclass, no significant differences were observed with the anti‐kappa IgG, suggesting equal coupling. Tenfold dilutions reduced this signal, particularly for IgG2, suggesting alternative configuration on the chip at lower densities (Fig. 1a). No anti‐kappa signal was detected when IgG were spotted at one RU. We then analysed the interaction between U937 cells with the IgG‐coupled chips. After injection of the U937 cells, they were allowed to interact with the sensor surface by sedimentation after stopping the flow (Fig. 1b). This resulted in specific interaction of the cells with IgG1, IgG3 and IgG4 compared to BSA, judged by the change in real‐time angle‐shift. We found that IgG3 was the most effective in triggering cell adherence, IgG1 and IgG4 were roughly half that effective, while IgG2 induced very little or no cell adherence. Interestingly, this preference of IgG3 was so pronounced that even at the 100‐fold dilution (∼1 nM) some of the spots gave a similar response as the 100 RU (Fig. 1c). Repeated measurements on a single chip indicated that quantitative measurements could be achieved between all spots within a single cycle, and between cycles for up to 14 independent runs after repeated use and regenerations (Supporting information, Fig. S1).

Figure 1.

Figure 1

Open in a new tab

U937 cells bind efficiently to human immunoglobulin (Ig) G1, IgG3 and IgG4, but not IgG2. (a) IgG subclasses, coupled equally, detected with kappa chain‐specific antibody. (b) Representative sensogram of IgG subclasses detected with U937 cells. (c) Cell binding expressed as percentage of the highest signal in each cycle. Data in (a,c) shows the mean of three replicate runs with three spots per run. Resonance unit values shown were recorded in the last 10 s of the 15 min incubation, marked with the dotted frame in (b). Bars represent means with error bars showing standard deviation.

Sera of RA patients lead to myeloid cell detection of VCP2

We next tested if serum‐derived reactivity towards antigens can also be detected using myeloid cells as readout. To this end, we compared the reactivity of sera from RA patients and normal healthy serum donors (NHS) towards the RA specific citrullinated MAPs, HCP2 and VCP2. As positive controls we spotted hIgG1 and IgG3 at 100 and 10 nM spotting concentrations, and BSA and PBS as negative controls. Incubation of sera showed typical binding resembling antibody–antigen (ligand‐receptor) during the association and dissociation phase (left and middle sides of sensograms in Fig. 2a, respectively, as summarized in the cartoon in Fig. 2b). We noted that sera of RA patients showed significantly elevated serum reactivity towards IgG1, consistent with increased levels of RF in these patients (right side, Fig. 2a,c). Similarly, sera of RA patients showed a significantly increased serum response to HCP2 and VCP2 compared to NHS patients (Fig. 2a,d). We then introduced the U937 cells, allowing them to interact with the immune complexes deposited on the sensor surface (right half of sensograms in Fig. 2a as summarized in Fig. 2b). U937 cells reacted equally strongly to IgG1‐ and IgG3‐equipped control spots treated with NHS and RA sera (Fig. 2a,e). Although many NHS showed elevated serum binding to HCP2, these did not result in myeloid U937‐binding (Fig. 2a,d–f). Importantly, U937 cells reacted significantly more strongly with RA serum‐treated VCP2 spots compared to NHS (Fig. 2f). The cellular response to HCP2 was not elevated significantly compared to NHS, although RA samples tended to give a stronger response. While for HCP2 no correlation was found between levels of serum and cell response, for VCP2 the cellular response correlated significantly with the level of serum response measured in the same run, showing moderate association (Fig. 2g).

Figure 2.

Figure 2

Open in a new tab

Comparison of healthy serum donors (NHS) and rheumatoid arthritis (RA) sera reactivity towards immunoglobulin (Ig) G multiple antigenic peptides (MAPs) and subsequent recognition by U937 cells. (a) Representative sensograms recorded for NHS and RA samples, on various ligands, showing phases of the measurement (depicted in (b)], with dashed frames indicating the response values evaluated. (c) Results of serum association measured on IgG1 and IgG3, and (d) MAPs: histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2), and (e–f) the following cell binding response on these ligands are shown. Symbols in (c–f) mark the mean response unit obtained on the ligand subtracted by phosphate‐buffered saline (PBS) background. Statistical significance (c–f) was calculated by Mann–Whitney U‐test. (g) Comparison of serum and cell binding response on MAPs; here Spearman's rank correlation coefficients (r) were calculated.

VCP2‐specific antibody responses correlated with cellular responses

The ability of U937 cells to differentiate among patient sera raised the question of whether it was associated with their antigen‐specific antibody isotype and subclass composition. For both HCP2 and VCP2, specific antibodies of multiple subclasses were detectable in most of the higher responding samples and the subclass levels themselves showed strong correlations with each other (Supporting information, Table S1). The same was true for the isotypes, with a strong significant correlation between the antigen‐specific IgA, IgG and IgM levels (Supporting information, Table S2). VCP2‐specific serum responses recorded on the IBIS chips correlated significantly with the IgG and IgA levels measured on antigen microarrays (see Supporting information) and with levels of IgG1, IgG3 and IgG4 measured with ELISA; however no correlations were found in the case of HCP2 (Supporting information, Fig. S2).

The antigen‐specific IgG and IgA levels showed significant correlation with cellular response to VCP2, but not with IgM (Fig. 3a). Looking closer at the IgG subclass responses, we found a strong significant correlation between IgG3 levels with the U937 reactivity to serum opsonized VCP2‐sensor chips (Fig. 3b). No significant correlations were found between antibody levels and U937 reactivity for HCP2 (Fig. 3a,b).

Figure 3.

Figure 3

Open in a new tab

Immunoglobulin levels against citrullinated peptides correlate with U937 responses in RA patients. (a) Histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2)‐specific immunoglobulin (Ig) A, IgG and IgM measured by microarrays and (b) IgG subclasses as determined by enzyme‐linked immunosorbent assay (ELISA) from rheumatoid arthritis (RA) samples were compared to cell response values obtained from the biosensor experiments measured on the same antigens. Symbols represent the mean of replicate measurements after subtraction of background values. Cell response on MAPs was expressed in ratio of cell response on hIgG1 10mM spots in the same cycle. Spearman's rank correlation coefficients (r) were calculated to evaluate the correlations.

Cell response on VCP2 shows significant association with CCP2 positivity and RA diagnosis

To evaluate its diagnostic performance, we compared the cellular responses towards the opsonized MAP biosensor with serological and clinical diagnostic RA criteria. For comparison of cellular responses, we categorized samples into positive and negative sets based on cell response results, using the highest measured cell response value in the NHS group as a cut‐off value. We then compared the cellular response to the results of the CCP2 ELISA. While VCP2 cell responses showed significant association in both comparisons (Table 1), no significant association was observed for HCP2. By measuring cell responses to the serum‐opsonized VCP2 chip, we could identify 11 of 13 seropositive (CCP2+) and 67% of clinically established RA patients.

Table 1.

Rheumatoid arthritis (RA) diagnosis and cyclic citrullinated peptide 2 (CCP2) positivity shows association with cellular response against viral citrullinated peptide 2 (VCP2)

U937 cell response
VCP2 10 mM HCP2 1 mM
+ – + –
CCP2 + 11 2 5 8
– 1 9 2 8
P = 0·0006** P = 0·4050, n.s.
RA + 12 6 7 11
– 0 5 0 5
P = 0·0137* P = 0·2719, n.s.
Open in a new tab

Diagnostic performance of cell binding response measured on MAPs after serum incubation with NHS or RA samples were compared to CCP2 tests and RA diagnosis. Results are summarized as a contingency table, separately for comparison with CCP2 test and RA diagnosis results. To assess the associations, Fisher's exact test was calculated. n.s. = not significant.

Discussion

In this work we examined the application of monocytic cell line U937 to detect immune complexes using label‐free SPRi in real time as a potential method to evaluate the clinical relevance of humoral autoantibody formation. We found that immune complexes are formed on relevant RA antigens spotted on the biosensor surface, which are recognized subsequently by myeloid cells. The magnitude of the cellular recognition seemed to be associated with the quality of the deposited antibodies, and to be related to clinical signatures in RA.

Detection of immune complexes using cells has several advantages beyond serology. This is because serology alone does not provide any information on the quality of the immune response besides isotype or subclass. Any additive qualitative information that affects recognition by effector cells is lost. This can be due to affinity of the antibodies involved, stoichiometry or antibody Fc‐glycosylation – all factors affecting binding to FcγR 5, 33, 34, 35.

Using a label‐free biosensor method for this task eliminates the need of excessive sample handling in the form of cellular labelling. By applying the antigens on a surface, as opposed to in solution, we potentially mimic surface exposed antigens which change the pathogenic autoantibody and Fc‐receptor interactions from low‐affinity and low‐avidity to high‐avidity interactions. This mimics the recognition of surface‐deposited antibodies – recognized more easily by myeloid cells – which may also occur in vivo. However, using SPR for this application has not been applied widely so far due to several challenges not solved easily with most currently available SPR equipment. Here, we utilized a system with reversed optics, allowing for sedimentation of the cells towards the sensor surface, equipped with up to 96 different antigens which can be monitored in parallel. During the process, sera and cells are injected serially. After injection of the cells, they are allowed to sediment onto the sensor surface and then interact with molecules opsonized on the sensor surface, allowing adhesion of the cells, registered as change in the angle shift measured by classical SPR methods 16.

First we looked at the cell adhesion to human IgG subclasses and found that the cells recognize and adhere to these molecules, presumably through their FcγR. U937 cells express FcγRs: FcγRIIa and FcγRI 36. The high‐affinity FcγRI can bind IgG1, IgG3 and IgG4, but not IgG2. U937 cells express the R131 variant of FcγRIIa 37, which binds IgG1 and IgG3 most efficiently, slightly better than IgG4, and has minimal reactivity to IgG2 12, 38. In addition, U937 cells may express the inhibitory FcγRIIb 39, 40, but this is still controversial 41. FcγRIIb has the lowest affinity to IgG of all human FcγRs and binds all subclasses except IgG2 12. Binding of U937 cells to human IgG subclasses in SPRi measurements followed the pattern of the relative affinity of FcγRs expressed on U937 cells, favouring IgG3 over IgG1 and IgG4 and showing no or little reactivity to IgG2. This is in accordance with our previous results based on nitrocellulose‐based microarray experiments 10.

Measurement of RA‐specific reactivity of sera by citrullin–arginin peptide pairs coupled to SPRi sensors have been demonstrated previously 29, also with HCP2 and VCP2 peptides in a Biacore system 42. In agreement with those studies, we also found that patients’ sera react to these peptides in our biosensor setup. We then injected U937 cells and allowed them to interact with the opsonized sensor surface without flow. We observed a specific interaction of the U937 cells with the MAPs‐sensor spots after opsonization with reactive sera on‐chip. Previous studies investigating anti‐keratin antibodies 43, and later ACPA in samples from RA patients, revealed different isotype distribution when measured on different peptides, mainly with high IgG1‐levels and frequent IgG4‐positivity, but without IgG2 43, 44, 45, 46, 47. Class‐switching is generally thought to develop before the onset of the specific spectra of symptoms characterizing RA 48. Comparison of ACPA isotypes in samples from RA patients and their healthy relatives showed that unaffected relatives may also have ACPA, most frequently IgG1 and IgA, while a more diverse isotype contribution was characteristic for RA symptoms 49. The presence of five or more ACPA antibody types and high levels of total IgG anti‐CCP2 were associated with more severe disease progression 44. In general IgM, IgA and the IgG subclasses all seem to contribute to ACPA‐mediated clinical symptoms, with the available data suggesting ACPA IgG being the most prominent disease inducer 44, 50, 51. In agreement with this published literature, it was difficult to pinpoint a single isotype or IgG‐subclass promoting the strongest cellular response, as all show underlying secondary correlations. Serum response measured on HCP2 discriminated the RA and NHS groups significantly, yet this did not lead to a significant cell response. This may be due possibly to high concentrations of low‐affinity IgM outcompeting IgG. The deposited IgM would also not be expected to lead to cellular activation, as no receptors for IgM have been described on U937 cells. This lack of correlation between serum response and antigen‐specific IgM results possibly from removal of low‐affinity IgM with thorough washing in our microarray protocol. However, VCP2‐specific IgA, IgG and IgG3 levels showed strong correlations with cellular response, mirroring reported clinical associations 46, 52, 53. For VCP2, serum and cell response showed a significant correlation which was not found for HCP2, suggesting a qualitatively distinct set of antibodies recognizing these two antigens. The moderate association in the case of VCP2 further supports the notion that cellular response, given a sufficient serum response, is driven rather by the quality over the quantity of the reacting serum components.

The present method shows that interactions of monocytoid cells with citrullinated peptides after incubation with patient sera is associated strongly with the diagnosis of RA and CCP positivity. Further development of the presented method, utilizing more target peptides and proteins in a single run, should help us to determine antigen‐specific immune response in more detail. Added information would be acquired if the donors’ own cells could be used, as the response is also likely to be influenced by the polymorphism of FcγRs 38, 54. Characterizing the effector function‐triggered cell activation by serum antibodies on different antigens could provide further insight into disease state and activity in a personalized manner. In conclusion, this method may detect those endowed with a pathogenic potential among disease‐specific antibodies, but further studies on a group of well‐characterized RA patients will be necessary to verify the clinical significance of these findings.

Disclosure

P. M. and P. R. are founders of Toscana Biomarkers Srl, the company that owns patent rights on VCP and HCP peptides. L. V. received reimbursement for attending a conference and regular payment for sample and data collection for this study from GAPAID project fund of University of Pécs.

Supporting information

Additional Supporting information may be found in the online version of this article at the publisher's web‐site:

Fig. S1. Repeated measurements of human immunoglobulin (hIg) G1 and hIgG3 with U937 cells. Cell adherence to triplicates of 100 nM spots of IgG1 and IgG3 was recorded in 22 cycles on the same sensor after each other. Each symbol shows results of a single measurement in a cycle, and bars mark the mean of the triplicates. Response expressed in resonance units (RU).

Click here for additional data file. (213.9KB, pdf)

Fig. S2. Correlation of antigen‐specific immunoglobulin levels with serum response on multiple antigenic peptides (MAPs): histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide (VCP2). (a) Comparison of antigen‐specific antibody levels measured by antigen microarray for immunoglobulin (Ig) G, IgA and IgM and (b) by enzyme‐linked immunosorbent assay (ELISA) for IgG subclasses with serum association response on MAPs. Spearman's rank correlation coe1fficients (r) were considered significant, as follows: *P < 0·05; **P < 0·01; n.s. = non‐significant. RU = response unit; RFI = relative fluorescent intensity; AU = arbitrary unit.

Click here for additional data file. (495KB, pdf)

Table S1. Correlation of antigen‐specific immunoglobulin (Ig) G subclass levels of rheumatoid arthritis (RA) serum samples, measured on histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Antigen‐specific IgG1–4 subclass levels were determined by enzyme‐linked immunosorbent assay (ELISA). Spearman's rank correlation coefficients (r) are shown in each cell with the row and column headers indicating the compared subclasses for VCP2 in dark grey, and in light grey for HCP2. Correlations were considered significant at P < 0·05.

Click here for additional data file. (342KB, pdf)

Table S2. Correlation of antigen‐specific immunoglobulin (Ig) G, IgM and IgA levels of rheumatoid arthritis (RA) serum samples, measured on histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Antigen‐specific levels of IgA, IgG and IgM of the samples were determined by antigen microarray. Spearman's rank correlation coefficients (r) are shown in each cell with the row and column headers indicating the compared isotypes for VCP2 in dark grey, and in light grey for HCP2. Correlations were considered significant at P < 0·05.

Click here for additional data file. (355.6KB, pdf)

Supporting Information Methods

Click here for additional data file. (251.1KB, pdf)

Acknowledgements

This work was supported by the European Union Seventh Framework Programme FP7/2007–2013 under grant agreement [GAPAID‐314971, FP7‐SME‐2012], entitled ‘Genes and proteins for autoimmunity diagnostics’. Support from the National Research, Development and Innovation Office – NKFIH to J. P., grant number K109683 is acknowledged. Z. S. was supported by an EFIS‐IL Short Term Fellowship.

References

  • 1. Gan SD, Patel KR. Enzyme immunoassay and enzyme‐linked immunosorbent assay. J Invest Dermatol 2013; 133:e12. [DOI] [PubMed] [Google Scholar]
  • 2. Willitzki A, Hiemann R, Peters V et al New platform technology for comprehensive serological diagnostics of autoimmune diseases. Clin Dev Immunol 2012; 2012:284740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 2007; 25:21–50.] [DOI] [PubMed] [Google Scholar]
  • 4. Kapur R, Kustiawan I, Vestrheim A et al A prominent lack of IgG1‐Fc fucosylation of platelet alloantibodies in pregnancy. Blood 2014; 123:471–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: from structure to effector functions. Front Immunol 2014; 5:520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Degn SE, Thiel S. Humoral pattern recognition and the complement system. Scand J Immunol 2013; 78:181–93. [DOI] [PubMed] [Google Scholar]
  • 7. Mantovani A, Valentino S, Gentile S, Inforzato A, Bottazzi B, Garlanda C. The long pentraxin PTX3: a paradigm for humoral pattern recognition molecules. Ann NY Acad Sci 2013; 1285:1–14. [DOI] [PubMed] [Google Scholar]
  • 8. Rojko JL, Evans MG, Price SA et al Formation, clearance, deposition, pathogenicity, and identification of biopharmaceutical‐related immune complexes: review and case studies. Toxicol Pathol 2014; 42:725–64. [DOI] [PubMed] [Google Scholar]
  • 9. Sundstrom C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U‐937). Int J Cancer 1976; 17:565–77. [DOI] [PubMed] [Google Scholar]
  • 10. Szittner Z, Papp K, Sandor N, Bajtay Z, Prechl J. Application of fluorescent monocytes for probing immune complexes on antigen microarrays. PLoS One 2013; 8:e72401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Boltz‐Nitulescu G, Willheim M, Spittler A, Leutmezer F, Tempfer C, Winkler S. Modulation of IgA, IgE, and IgG Fc receptor expression on human mononuclear phagocytes by 1 alpha,25‐dihydroxyvitamin D3 and cytokines. J Leukoc Biol 1995; 58:256–62. [DOI] [PubMed] [Google Scholar]
  • 12. Bruhns P, Iannascoli B, England P et al Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 2009; 113:3716–25. [DOI] [PubMed] [Google Scholar]
  • 13. Yanase Y, Hiragun T, Ishii K et al Surface plasmon resonance for cell‐based clinical diagnosis. Sensors (Basel) 2014; 14:4948–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Suraniti E, Sollier E, Calemczuk R et al Real‐time detection of lymphocytes binding on an antibody chip using SPR imaging. Lab Chip 2007; 7:1206–8. [DOI] [PubMed] [Google Scholar]
  • 15. Quinn JG, O'Kennedy R, Smyth M, Moulds J, Frame T. Detection of blood group antigens utilising immobilised antibodies and surface plasmon resonance. J Immunol Methods 1997; 206:87–96. [DOI] [PubMed] [Google Scholar]
  • 16. Schasfoort RB, Bentlage AE, Stojanovic I et al Label‐free cell profiling. Anal Biochem 2013; 439:4–6. [DOI] [PubMed] [Google Scholar]
  • 17. Stojanovic I, Schasfoort RB, Terstappen LW. Analysis of cell surface antigens by Surface Plasmon Resonance imaging. Biosens Bioelectron 2014; 52:36–43. [DOI] [PubMed] [Google Scholar]
  • 18. Yanase Y, Hiragun T, Yanase T, Kawaguchi T, Ishii K, Hide M. Application of SPR imaging sensor for detection of individual living cell reactions and clinical diagnosis of type I allergy. Allergol Int 2013; 62:163–9. [DOI] [PubMed] [Google Scholar]
  • 19. Peterson AW, Halter M, Tona A, Bhadriraju K, Plant AL. Using surface plasmon resonance imaging to probe dynamic interactions between cells and extracellular matrix. Cytometry A 2010; 77:895–903. [DOI] [PubMed] [Google Scholar]
  • 20. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. New Engl J Med 2011; 365:2205–19. [DOI] [PubMed] [Google Scholar]
  • 21. Nishimura K, Sugiyama D, Kogata Y et al Meta‐analysis: diagnostic accuracy of anti‐cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann Intern Med 2007; 146:797–808. [DOI] [PubMed] [Google Scholar]
  • 22. Nielsen SF, Bojesen SE, Schnohr P, Nordestgaard BG. Elevated rheumatoid factor and long term risk of rheumatoid arthritis: a prospective cohort study. BMJ 2012; 345:e5244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Soltys AJ, Hay FC, Bond A et al The binding of synovial tissue‐derived human monoclonal immunoglobulin M rheumatoid factor to immunoglobulin G preparations of differing galactose content. Scand J Immunol 1994; 40:135–43. [DOI] [PubMed] [Google Scholar]
  • 24. Willemze A, Trouw LA, Toes RE, Huizinga TW. The influence of ACPA status and characteristics on the course of RA. Nat Rev Rheumatol 2012; 8:144–52. [DOI] [PubMed] [Google Scholar]
  • 25. Scherer HU, van der Woude D, Ioan‐Facsinay A et al Glycan profiling of anti‐citrullinated protein antibodies isolated from human serum and synovial fluid. Arthritis Rheum 2010; 62:1620–9. [DOI] [PubMed] [Google Scholar]
  • 26. Aletaha D, Neogi T, Silman AJ et al 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62:2569–81. [DOI] [PubMed] [Google Scholar]
  • 27. Pruijn GJ, Wiik A, van Venrooij WJ. The use of citrullinated peptides and proteins for the diagnosis of rheumatoid arthritis. Arthritis Res Ther 2010; 12:203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Clavel C, Nogueira L, Laurent L et al Induction of macrophage secretion of tumor necrosis factor alpha through Fcgamma receptor IIa engagement by rheumatoid arthritis‐specific autoantibodies to citrullinated proteins complexed with fibrinogen. Arthritis Rheum 2008; 58:678–88. [DOI] [PubMed] [Google Scholar]
  • 29. Lokate AM, Beusink JB, Besselink GA, Pruijn GJ, Schasfoort RB. Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging. J Am Chem Soc 2007; 129:14013–8. [DOI] [PubMed] [Google Scholar]
  • 30. Pratesi F, Dioni I, Tommasi C et al Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps. Ann Rheum Dis 2014; 73:1414–22. [DOI] [PubMed] [Google Scholar]
  • 31. Pratesi F, Tommasi C, Anzilotti C et al Antibodies to a new viral citrullinated peptide, VCP2: fine specificity and correlation with anti‐cyclic citrullinated peptide (CCP) and anti‐VCP1 antibodies. Clin Exp Immunol 2011; 164:337–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Kapur R, Heitink‐Polle KM, Porcelijn L et al C‐reactive protein enhances IgG‐mediated phagocyte responses and thrombocytopenia. Blood 2015; 125:1793–802. [DOI] [PubMed] [Google Scholar]
  • 33. Diebolder CA, Beurskens FJ, de Jong RN et al Complement is activated by IgG hexamers assembled at the cell surface. Science 2014; 343:1260–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Lux A, Yu X, Scanlan CN, Nimmerjahn F. Impact of immune complex size and glycosylation on IgG binding to human FcgammaRs. J Immunol 2013; 190:4315–23. [DOI] [PubMed] [Google Scholar]
  • 35. Kapur R, Einarsdottir HK, Vidarsson G. IgG‐effector functions: ‘the good, the bad and the ugly’. Immunol Lett 2014; 160:139–44. [DOI] [PubMed] [Google Scholar]
  • 36. Tacken PJ, Batenburg JJ. Monocyte CD64 or CD89 targeting by surfactant protein D/anti‐Fc receptor mediates bacterial uptake. Immunology 2006; 117:494–501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Valenzuela NM, Trinh KR, Mulder A, Morrison SL, Reed EF. Monocyte recruitment by HLA IgG‐activated endothelium: the relationship between IgG subclass and FcgammaRIIa polymorphisms. Am J Transplant 2015; 15:1502–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Hogarth PM, Pietersz GA. Fc receptor‐targeted therapies for the treatment of inflammation, cancer and beyond. Nat Rev Drug Discov 2012; 11:311–31. [DOI] [PubMed] [Google Scholar]
  • 39. Valenzuela NM, Mulder A, Reed EF. HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P‐selectin and, depending on subclass, by engaging FcgammaRs. J Immunol 2013; 190:6635–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Tridandapani S, Siefker K, Teillaud JL, Carter JE, Wewers MD, Anderson CL. Regulated expression and inhibitory function of Fcgamma RIIb in human monocytic cells. J Biol Chem 2002; 277:5082–9. [DOI] [PubMed] [Google Scholar]
  • 41. Veri MC, Gorlatov S, Li H et al Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma‐receptor IIB (CD32B) from the activating Fcgamma‐receptor IIA (CD32A): biochemical, biological and functional characterization. Immunology 2007; 121:392–404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Rossi G, Real‐Fernandez F, Panza F et al Biosensor analysis of anti‐citrullinated protein/peptide antibody affinity. Anal Biochem 2014; 465:96–101. [DOI] [PubMed] [Google Scholar]
  • 43. Vincent C, Serre G, Basile JP et al Subclass distribution of IgG antibodies to the rat oesophagus stratum corneum (so‐called anti‐keratin antibodies) in rheumatoid arthritis. Clin Exp Immunol 1990; 81:83–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. van der Woude D, Syversen SW, van der Voort EI et al The ACPA isotype profile reflects long‐term radiographic progression in rheumatoid arthritis. Ann Rheum Dis 2010; 69:1110–6. [DOI] [PubMed] [Google Scholar]
  • 45. Chapuy‐Regaud S, Nogueira L, Clavel C, Sebbag M, Vincent C, Serre G. IgG subclass distribution of the rheumatoid arthritis‐specific autoantibodies to citrullinated fibrin. Clin Exp Immunol 2005; 139:542–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Verpoort KN, Jol‐van der Zijde CM, Papendrecht‐van der Voort EA et al Isotype distribution of anti‐cyclic citrullinated peptide antibodies in undifferentiated arthritis and rheumatoid arthritis reflects an ongoing immune response. Arthritis Rheum 2006; 54:3799–808. [DOI] [PubMed] [Google Scholar]
  • 47. Panza F, Pratesi F, Valoriani D, Migliorini P. Immunoglobulin G subclass profile of anticitrullinated peptide antibodies specific for Epstein Barr virus‐derived and histone‐derived citrullinated peptides. J Rheumatol 2014; 41:407–8. [DOI] [PubMed] [Google Scholar]
  • 48. van Venrooij WJ, van Beers JJ, Pruijn GJ. Anti‐CCP antibodies: the past, the present and the future. Nat Rev Rheumatol 2011; 7:391–8. [DOI] [PubMed] [Google Scholar]
  • 49. Ioan‐Facsinay A, Willemze A, Robinson DB et al Marked differences in fine specificity and isotype usage of the anti‐citrullinated protein antibody in health and disease. Arthritis Rheum 2008; 58:3000–8. [DOI] [PubMed] [Google Scholar]
  • 50. Ossipova E, Cerqueira CF, Reed E et al Affinity purified anti‐citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint. Arthritis Res Ther 2014; 16:R167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Harre U, Georgess D, Bang H et al Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest 2012; 122:1791–802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Shiozawa K, Kawasaki Y, Yamane T et al Anticitrullinated protein antibody, but not its titer, is a predictor of radiographic progression and disease activity in rheumatoid arthritis. J Rheumatol 2012; 39:694–700. [DOI] [PubMed] [Google Scholar]
  • 53. Demoruelle MK, Deane K. Antibodies to citrullinated protein antigens (ACPAs): clinical and pathophysiologic significance. Curr Rheumatol Rep 2011; 13:421–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54. Li X, Gibson AW, Kimberly RP. Human FcR polymorphism and disease. Curr Top Microbiol Immunol 2014; 382:275–302. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting information may be found in the online version of this article at the publisher's web‐site:

Fig. S1. Repeated measurements of human immunoglobulin (hIg) G1 and hIgG3 with U937 cells. Cell adherence to triplicates of 100 nM spots of IgG1 and IgG3 was recorded in 22 cycles on the same sensor after each other. Each symbol shows results of a single measurement in a cycle, and bars mark the mean of the triplicates. Response expressed in resonance units (RU).

Click here for additional data file. (213.9KB, pdf)

Fig. S2. Correlation of antigen‐specific immunoglobulin levels with serum response on multiple antigenic peptides (MAPs): histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide (VCP2). (a) Comparison of antigen‐specific antibody levels measured by antigen microarray for immunoglobulin (Ig) G, IgA and IgM and (b) by enzyme‐linked immunosorbent assay (ELISA) for IgG subclasses with serum association response on MAPs. Spearman's rank correlation coe1fficients (r) were considered significant, as follows: *P < 0·05; **P < 0·01; n.s. = non‐significant. RU = response unit; RFI = relative fluorescent intensity; AU = arbitrary unit.

Click here for additional data file. (495KB, pdf)

Table S1. Correlation of antigen‐specific immunoglobulin (Ig) G subclass levels of rheumatoid arthritis (RA) serum samples, measured on histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Antigen‐specific IgG1–4 subclass levels were determined by enzyme‐linked immunosorbent assay (ELISA). Spearman's rank correlation coefficients (r) are shown in each cell with the row and column headers indicating the compared subclasses for VCP2 in dark grey, and in light grey for HCP2. Correlations were considered significant at P < 0·05.

Click here for additional data file. (342KB, pdf)

Table S2. Correlation of antigen‐specific immunoglobulin (Ig) G, IgM and IgA levels of rheumatoid arthritis (RA) serum samples, measured on histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Antigen‐specific levels of IgA, IgG and IgM of the samples were determined by antigen microarray. Spearman's rank correlation coefficients (r) are shown in each cell with the row and column headers indicating the compared isotypes for VCP2 in dark grey, and in light grey for HCP2. Correlations were considered significant at P < 0·05.

Click here for additional data file. (355.6KB, pdf)

Supporting Information Methods

Click here for additional data file. (251.1KB, pdf)

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES