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.

Alpha-2-Macroglobulin in Inflammation, Immunity and Infections

“…In a recent study, A2M was also identified as the possible antigen causing hexamerization/aggregation of IgG as seen in patients with chronic lymphocytic leukemia and chronic activation of the complement classical pathway. A2M was found to be part of the IgG hexamer complex and present at the cell surface of malignant B lymphocytes through binding with GRP78 ( 36 ). The mechanism through which such interaction would occur remains to be determined.…”

Increased serum level of alpha-2 macroglobulin and its production by B-lymphocytes in chronic lymphocytic leukemia

“…Our previous studies that focused on the chronic CP activation in CLL included characterization of the cell-free IgG-aggregates that exist in CLL serum and act as a CP activator (4,16). In the initiation of CP activation, IgGhexamers bind C1, the first component of the CP, and activate a cascade of events until C5b-9 (MAC), the final complement Correlation of the serum A2M levels with complement components.…”

“…This is particularly evident at advanced CLL stages. Although some of the A2M molecules bind to T-lymphocytes and to the B-lymphocytes via the CD91 receptor (24) and the GRP78 receptor (4,25,26), respectively, the increased amounts of A2M produced, yield serum levels that correlate very well with the lymphocytes' counts in the circulation. In the experiments assessing A2M production by B-cells, the potential binding to the CD91 receptor on Tlymphocytes is irrelevant, since these experiments were performed using only B-cells (in case of cell lines) or 99% pure primary B-cells preparations.…”

“…Similar to other members of the thioester-containing proteins (TEPs) family, named after their active site that functions by forming covalent bonds with specific molecular targets, A2M plays an important role in clearance of proteases from the circulation, regulation of fibrinolysis, coagulation and complement activation (3). A2M also participates, with IgG molecules, in formation of immune complexes that generate aggregated/hexameric structures (A2M-IgG-hexamers) and activate the classical pathway (CP) of the complement system in patients with Chronic Lymphocytic Leukemia (CLL) (4).…”

“…It affects the B-type lymphocytes in the bone marrow in 95% of the patients, and is characterized by increased numbers of monoclonal Blymphocytes (>5x10 3 /µl) that express specific antigens (CD5, CD19, CD20, CD23) on their surface. A2M is involved in the chronic CP activation in CLL (4), which decreases the CP activity in these patients. This decrease in CP activity compromises the CP dependent mechanisms activated by the immunotherapeutic drugs, such as the complement dependent cytotoxicity (CDC), and may eventually affect the outcomes of the immunotherapy.…”