Pro-inflammatory State in Monoclonal Gammopathy of Undetermined Significance and in Multiple Myeloma Is Characterized by Low Sialylation of Pathogen-Specific and Other Monoclonal Immunoglobulins

Abstract

Multiple myeloma (MM) and its pre-cancerous stage monoclonal gammopathy of undetermined significance (MGUS) allow to study immune responses and the chronology of inflammation in the context of blood malignancies. Both diseases are characterized by the production of a monoclonal immunoglobulin (mc Ig) which for subsets of MGUS and MM patients targets pathogens known to cause latent infection, a major cause of inflammation. Inflammation may influence the structure of both polyclonal (pc) Ig and mc Ig produced by malignant plasma cells via the sialylation of Ig Fc fragment. Here, we characterized the sialylation of purified mc and pc IgGs from 148 MGUS and MM patients, in comparison to pc IgGs from 46 healthy volunteers. The inflammatory state of patients was assessed by the quantification in serum of 40 inflammation-linked cytokines, using Luminex technology. While pc IgGs from MGUS and MM patients showed heterogeneity in sialylation level, mc IgGs from both MGUS and MM patients exhibited a very low level of sialylation. Furthermore, mc IgGs from MM patients were less sialylated than mc IgGs from MGUS patients (p < 0.01), and mc IgGs found to target an infectious pathogen showed a lower level of sialylation than mc IgGs of undetermined specificity (p = 0.048). Regarding inflammation, 14 cytokines were similarly elevated with a p value < 0.0001 in MGUS and in MM compared to healthy controls. MM differed from MGUS by higher levels of HGF, IL-11, RANTES and SDF-1-α (p < 0.05). MGUS and MM patients presenting with hyposialylated pc IgGs had significantly higher levels of HGF, IL-6, tumor necrosis factor-α, TGF-β1, IL-17, and IL-33 compared to patients with hyper-sialylated pc IgGs (p < 0.05). In MGUS and in MM, the degree of sialylation of mc and pc IgGs and the levels of four cytokines important for the anti-microbial response were correlated, either positively (IFN-α2, IL-13) or negatively (IL-17, IL-33). Thus in MGUS as in MM, hyposialylation of mc IgGs is concomitant with increased levels of cytokines that play a major role in inflammation and anti-microbial response, which implies that infection, inflammation, and abnormal immune response contribute to the pathogenesis of MGUS and MM.

Keywords: cytokines; immunoglobulin G sialylation; infection; inflammation; monoclonal gammopathy of undetermined significance; monoclonal immunoglobulin; myeloma.

Figure 1

Purification scheme of mc IgGs from the serum of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. (A) Mc and pc IgGs from MGUS and MM patients were submitted to high-resolution agarose gel electrophoresis and then cut from the gel. (B) The purity of mc and pc IgGs was checked via isoelectro-focalization (IEF) and immunoblotting using a peroxydase coupled to an anti-human IgG-γ chain antibody (HRP anti-human IgG Ab). (C) The sialylation of mc IgG was assessed after incubation with biotinylated Sambucus nigra agglutinin (SNA) and streptavidin peroxidase. (B,C) are representative of purified basic or acid mc IgG, respectively.

Figure 2

Sialylation level of IgGs in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). (A) Sialylation level of purified pc IgGs from healthy volunteers (n = 46) and myeloproliferative neoplasms (n = 40), and from purified mc IgGs (n = 148) and pc IgGs (n = 142) from MGUS and SM/MM patients (for 6 patients, pc IgGs could not be purified). Percentages indicate the % of patients whose IgGs present a low (<0.5), normal (0.5–1.5), of high (>1.5) level of sialylation. (B) Degree of sialylation of purified pc IgGs and mc IgGs from MGUS (n = 68) and SM/MM (n = 80) patients. Sialylation level of purified pc IgGs (C) and mc IgGs (D) from MM patients with pathogen-specific mc IgG (MIAA+ patients) were then compared with pc and mc IgGs from other MM patients. Bars indicate means ± SEM. Statistical analysis was performed using one-way ANOVA test followed by Tukey’s Multiple Comparison Test for (A), unpaired t-test for (B) and (C), and unpaired t-test with Welch’s correction for (D). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Sialylation of IgGs as assessed by mass spectrometry: Purified pc IgGs from six healthy volunteers (HV) and pc IgGs and mc IgGs from eight patients [four monoclonal gammopathy of undetermined significance (MGUS) and four multiple myeloma (MM)] were analyzed by mass spectrometry. The percentage of sialylated glycoforms (G1FS + G2FS/total peaks) was determined in pc and mc IgG fractions. Bars indicate means ± SEM. Statistical analysis was performed using one-way ANOVA test after a Kolmogorov–Smirnov normality test followed by Tukey’s comparison test (^*1p < 0.05); and a paired t-test for pc IgG and mc IgG from MGUS and MM patients (^*2p < 0.05).

Figure 4

Cytokine levels in the serum of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. Forty cytokines and 2 cytokine receptors were quantified using the Biorad Luminex technology in the serum of 34 MGUS and 30 MM patients. The 6 cytokines found to be differently expressed in MGUS vs MM patients were: IL-11, HGF, SDF-1α, RANTES, TFG-β₁, TFG-β₂, and TGF-β₃. Horizontal bars indicate median values ± ranges. Statistical analysis was performed using Student t-test. *p < 0.05 and **p < 0.01.

Figure 5

Correlations between cytokine levels and the concentration of β₂-microglobulin in serum of multiple myeloma (MM) patients. β₂-microglobulin concentration was found to be positively correlated with the concentration of IL-9, IL-26, MIP-1β, and PDGF-BB for the 19 MM patients with available β₂-microglobulin data. Statistical analysis was performed using the Spearman t-test.

Figure 6

Cytokine levels in the serum of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SM)/Multiple myeloma (MM) patients with hyposialylated or hyper-sialylated pc IgGs. Forty cytokines and 2 cytokine receptors were quantified using the Biorad Luminex technology in the serum of 19 MGUS or SM/MM patients with hyposialylated pc IgGs (hypo), and 16 MGUS or SM/MM patients with hyper-sialylated pc IgGs (hyper). The 6 cytokines found to be more expressed in patients with hyposialylated pcIgGs were interleukin 6 (IL-6), hepatocyte growth factor (HGF), tumor necrosis factor (TNF)-α, TGF-β₁, IL-17, and IL-33. Horizontal bars indicate median values ± ranges. Statistical analysis was performed using Mann–Whitney U test. *p < 0.05, **p < 0.01, and ***p < 0.001. Normal values for IL-6: <9 pg/mL; HGF, median: 195 pg/mL, range: 63–1,283 pg/mL; TNF-α, median: 0 pg/mL, range: 6–98 pg/mL; TGF-β₁, median: 47 pg/mL, range: 0–932 pg/mL; IL-17, median: 0 pg/mL, range: 0.22–31 pg/mL; and IL-33, not defined.

Figure 7

Correlations between cytokine levels in serum of multiple myeloma (MM) patients and level of IgG sialylation. (A) Pc IgGs sialylation level and the concentration of leptin (men only), IL-33, and IL-17 were negatively correlated (n = 25 MM patients with available pc IgG sialylation data). (B) Mc IgG sialylation level was negatively correlated with leptin (men only), and positively correlated with the concentration of interferon (IFN)-α₂ and IL-13 (n = 30 MM patients with available mc IgG sialylation data). Statistical analysis was performed using the Spearman t-test.

Figure 8

Sialylation level of mc IgG from patients diagnosed with multiple myeloma (MM) at Durie–Salmon Staging (DSS) I, II, or III. Sialylation level of mc IgGs was compared among groups of MM patients with DSS stage I, II, or III. Horizontal bars indicate mean ± SEM. Statistical analysis was performed using Kruskall–Wallis test followed by Dunn’s Multiple Comparison test. *p < 0.05.

Figure 9

Schematic model of the regulation loop linking inflammation and IgG sialylation in multiple myeloma (MM). A pro-inflammatory microenvironment induces the secretion by malignant plasma cells of high amount of hyposialylated mc IgG which preferentially bind to activating receptors FcγRs (ITAM, blue pathway). To the contrary, sialylated IgGs preferentially bind to the inhibiting receptor FcγRIIb (ITIM, red pathway). The activation of macrophages by hyposialylated IgGs leads to the secretion of pro-inflammatory cytokines TGF-β, interleukin 6 (IL-6), tumor necrosis factor (TNF)-α and IL-33. TGF-β, IL-6, and TNF-α are known to stimulate the expression of IL-17 by Th17-lymphocytes, which are elevated in MM. IL-17 may inhibit the expression of the α2,6-sialyltransferase thus contributing to decrease IgG sialylation. MM patients with pathogen-specific mc IgG presented with the less hyposialylated mc IgG and highest level of IL-22, IL-26 and IL-33 (green cytokines) suggesting that this regulation loop may be amplified.