The Role of Alpha 2 Macroglobulin in IgG-Aggregation and Chronic Activation of the Complement System in Patients With Chronic Lymphocytic Leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. One of the treatments offered for CLL is immunotherapy. These treatments activate various cellular and biochemical mechanisms, using the complement system. Recently it was shown that the complement system in CLL patients is persistently activated at a low level through the classical pathway (CP). The mechanism of chronic CP activation involves the formation of IgG-hexamers (IgG-aggregates). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur after antigen binding. The present study investigated the formation of IgG-hexamers in CLL patients and normal (non-malignant) controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and 12 NC were used for separation of IgG- aggregates. The obtained IgG- aggregates were measured and used for assessment of CP activation. For evaluation of the presence of IgG- aggregates on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG- aggregates were higher in CLL and they activated the complement system to a higher extent than in NC. Alpha 2 macroglobulin (A2M) was identified as the antigen causing the hexamerization/aggregation of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the cell surface. The exact time course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens.

Keywords: IgG-hexamers,; alpha 2 macroglobulin; chronic lymphocytic leukemia; classical pathway; complement system.

Figure 1

Cell free IgG-hexamers patients and NC subjects. IgG-hexamers were purified from sera/plasma of CLL patients and NC subjects. (A) The percentage of IgG-hexamers were calculated. Detectable Ig-C5a (Det.Ig-C5a), n = 23, undetectable Ig-C5a (Undet.Ig-C5a) n = 7, NC n = 12. *, ** indicates significant p values (p < 0.04, 0.004, respectively) vs NC. (B) The samples were studied by Western blot using anti-IgG antibodies (representative results), equal protein amounts (10µg) were loaded in each lane. In vitro aggregated commercial IgG (Agg.IgG) was separated in parallel as a control. The heavy and light chains of the IgG are indicated by arrows.

Figure 2

Activation of the complement system by IgG-hexamers. Complement activity was measured in normal serum after incubation with IgG-hexamers from NC and patients (A). Serum samples incubated with buffer were used as a negative control. Proteins that did not bind to the protein G columns (non-IgG proteins, ◊), and non-hexameric IgG (monomeric IgG, □) were used for complement activation in C1q depleted serum (B) and factor B depleted serum (C). Activation was followed by the levels of sC5b-9. Detectable Ig-C5a (Det.Ig-C5a) n = 12, Undetectable Ig-C5a (Undet.Ig-C5a) n = 6, NC n = 8. *, ** indicates significant p values (p < 0.05, 0.005, respectively) compared to NC and No-hexamers.

Figure 3

IgG-hexamers on B cell surface. Blood samples from CLL patients and NC were stained with fluorescent antibodies against CD45, CD19 and C1, and tested in a flow cytometer. representative results are shown (A, B). The results were gated on WBC (A, C) or on lymphocytes (B, D). Detectable Ig-C5a n = 11; Undetectable Ig-C5a n = 6; NC n = 8. **, *** indicate significant p values (p < 0.01, 0.001, respectively) compared to NC.

Figure 4

Separation of the IgG-hexamer samples. (A) Samples of NC (a, b), CLL patients (c–f) and commercial IgG (g, h) were separated and silver stained. Heavy (γ) and light chain are indicated by arrows. Additional proteins besides heavy and light chains are marked in frames. (B) The process of selection of the resulting sequence data included elimination of all the IgG-related sequences, low molecular mass peptides, sequences with a total number of identified peptide sequences (peptide spectrum matches-#PSMs)<30, and sequences with coverage<25.

Figure 5

A2M presence in IgG-hexamers samples. A2M was identified in IgG-hexamers samples by Western blot using anti-A2M antibodies (representative results). Purified commercial A2M, reduced (red.) and non-reduced (non.R) were used as a positive control.

Figure 6

Association of the A2M-IgG-hexamers with B lymphocytes. Blood samples from CLL patients and NC were stained with fluorescent antibodies against CD45, CD19, C1, A2M and GRP78 (Bip) and tested in a flow cytometer. Representative results are shown.

Figure 7

IgG-hexamer formation after in-vitro incubation of monomeric IgG with A2M. Monomeric IgG were incubated with purified commercial A2M, HSA or PBS and the generated IgG-hexamers were separated. IgG were quantified by ELISA and the percent of IgG-hexamers was calculated. *, ns indicate significant (< 0.05) and non-significant p values, respectively.