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

Abstract

Chronic lymphocytic leukemia (CLL), the most common adult's leukemia in the western world, is caused in 95% of the cases by uncontrolled proliferation of monoclonal B-lymphocytes. The complement system in CLL is chronically activated at a low level via the classical pathway (CP). This chronic activation is induced by IgG-hexamers, which are formed after binding to alpha-2-macroglobulin (A2M). The study investigated for the first time the serum levels of A2M in CLL patients, their association with the disease severity, and A2M production by the malignant B-lymphocytes. Blood samples were collected from 65 CLL patients and 30 normal controls (NC) subjects, and used for quantifications of the A2M levels, the complement activation marker (sC5b-9), the complement components C2, C3 and C4, and clinical biochemistry and hematology parameters. The production of A2M was studied in B-lymphocytes isolated from blood samples as well as in CLL and non-CLL cell lines.The serum A2M levels were significantly higher in CLL patients vs NCs, showing values of 3.62 ± 0.22 and 1.97 ± 0.10 mg/ml, respectively. Within the CLL group, A2M levels correlated significantly with the disease stage, with sC5b-9, and with clinical indicators of the disease severity. Increased A2M production was showed in three out of four CLL B-lymphocytic lines that were studied, as compared to non-CLL lines, to a non-lymphocytic line, and to blood-derived primary B-lymphocytes. A2M production was further increased both in primary cells and in the CLL cell-line after incubation with CLL sera, compared to NC sera. This study shows for the first time that serum A2M levels in CLL are significantly increased, likely due to A2M production by the malignant B-lymphocytes, and are correlated with the disease severity and with chronic complement activation. The moderate change in A2M production after incubation with NC sera in-vitro supports the hypothesis that inhibition of excess A2M production can be achieved, and that this may potentially down-regulate the IgG-hexamerization and the resulting chronic CP activation. This may also help restore complement system activity, and eventually improve complement activity and immunotherapy outcomes in CLL.

Keywords: B-lymphocytes; alpha-2-macroglobulin (A2M); chronic lymphocytic leukemia; classical pathway of complement; complement system.

Figure 1

Serum A2M levels in CLL patients and NC subjects. (A) A2M levels were determined (using ELISA) in sera of CLL patients (n = 65) and NC subjects (n = 30). **** indicates p<0.0001 by t-test. (B) A2M levels in sera of CLL patients divided according to the CLL stage (Binet staging; stage A: n=29, stage B: n=23, stage C: n=12). **** and ** indicate p<0.0001 and p<0.02, respectively, vs. each other group. (C) Serum A2M levels in CLL patients divided according to the mutation associated with CLL. * indicates p<0.05 vs. each other group, # indicates p<0.05 vs. the NCs and vs. the CLL groups with 11q- and trisomy 12, as determined by the Mann-Whitney test.

Figure 2

Correlation of the serum A2M levels with complement components. The levels of the complement components C2-C4 and the complement activity marker sC5b-9 (the final product of complement activation) were measured in patients’ sera. A2M levels were correlated with C2 (A), C3 (B), C4 (C) and sC5b-9 (D). The dashed line indicates 15 mg/dL, which is the lower level of the normal range (15-57 mg/dL).

Figure 3

A2M production in B-Cells. The production of A2M was studied in the CLL cell lines MEC-2, JVM-2, JVM-13, HG-3, in the non-CLL B-lymphocytic cell lines SU-DHL-4 and SU-DHL-5, and in one monocytic cell line, THP-1. The assay was repeated ≥3 times for each cell line (n = 5 for MEC-2 and HG-3, n = 4 for JVM-13). A2M production was also studied in B-lymphocytes (primary cells) separated from peripheral blood of CLL patients (CLL lym.; n = 4) and NC subjects (NC lym.; n = 4). Cells were incubated in triplicates at a density of 20,000-40,000 cells per 100µl in the appropriate medium for each line, for 12-72 hrs and the ΔA2M levels were calculated for 100,000 cells for 24 hrs. ** indicates significant p values < 0.01 compared to each of the other cell lines, except for JVM-2; * indicates significant p values < 0.05 compared to each of the other cell lines, except for MEC-2; # indicates significant p values (p< 0.05) compared to each of the other cell lines, except for JVM-13 and THP-1.

Figure 4

A2M production in B-Cells after incubation with sera. (A) The production of A2M was studied in the CLL cell line MEC-2 (n = 25) and in primary B-lymphocytes (B) that were separated from peripheral blood of CLL patients (CLL B-lym.) and NC subjects (NC B-lym.);. Cells were incubated with NC sera (n = 10 with MEC-2 cells, n=11 with NC-lym. and n = 12 with CLL lym.) or with CLL sera (n = 15 with MEC-2 cells, n = 12 with NC-lym. and n = 11 with CLL lym.), A2M levels were measured and the ΔA2M levels were calculated for 100,000 cells per 24 hrs. ** indicates significant p values < 0.01 and * indicates significant p values < 0.05.