Uremia-related oxidative stress in leukocytes is not triggered by β2-microglobulin
Nathalie Neirynck1, Griet Glorieux, Jente Boelaert
1Nephrology Division, Department of Internal Medicine, Ghent University Hospital, Gent, Belgium.
Insights
Beta-2 microglobulin (B2M) does not appear to directly cause vascular damage by inducing leukocyte oxidative burst. Contaminants in B2M preparations, not B2M itself, likely caused prior observed inflammatory effects.
Area of Science:
- Nephrology
- Immunology
- Cardiology
Background:
- Chronic kidney disease (CKD) is linked to inflammation and cardiovascular disease (CVD).
- Beta-2 microglobulin (B2M) is a potential biomarker for CVD outcomes in CKD patients.
- B2M's role in vascular disease pathogenesis, possibly via leukocyte activation, is under investigation.
Purpose of the Study:
- To investigate if beta-2 microglobulin (B2M) is proinflammatory.
- To determine if B2M induces oxidative burst in leukocytes.
Main Methods:
- Oxidative burst measured in healthy volunteers' whole blood stimulated with fMLP, E. coli, or PMA.
- Tested human B2M (hB2M) and purified dB2M, with and without dialysis, against saline and uremic whole blood.
- Assessed reactive oxygen species (ROS) in response to lipopolysaccharide (LPS) as a comparator.
Main Results:
- Unpurified hB2M significantly enhanced ROS in monocytes and granulocytes.
- Purified dB2M did not increase burst activity, indicating contamination was responsible for initial effects.
- Low endotoxin levels in hB2M (<1.5 EU/mL) did not induce oxidative stress.
Conclusions:
- B2M may not directly cause vascular damage by inducing leukocyte free radical production.
- Contaminants in B2M preparations can skew research findings on inflammatory responses.
- Further research should carefully exclude biases from non-LPS contaminants.
Background:
Chronic kidney disease (CKD) is characterized by low-grade inflammation and increased risk for cardiovascular disease. The interest in β2-microglobulin (B2M) as a marker for cardiovascular outcome with and without CKD has grown. Clinical studies suggested that B2M could be involved in the pathogenesis of vascular disease, for which chronic leukocyte activation is a pathogenic factor. We investigated whether B2M is proinflammatory by inducing oxidative burst in leukocytes.
Methods:
Oxidative burst was measured at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP), Escherichia coli, or phorbol-12-myristate-acetate (PMA) in the whole blood of healthy volunteers in the absence (saline) and presence of human B2M (hB2M; 10 and 50 mg/L) versus uremic whole blood. Because of suspicion of contamination, hB2M was dialyzed for purification and purified B2M (dB2M) and dialysates were tested in the burst test. As a comparator, reactive oxygen species (ROS) in response to lipopolysaccharide (LPS) was measured.
Results:
Unpurified hB2M strongly enhanced ROS in monocytes and granulocytes after E. coli and PMA and moderately after fMLP stimulation compared with control (P < .01) and uremia (P < .01) whereas at baseline hB2M only induced ROS in granulocytes (P < .05). After purification, dB2M no longer increased burst activity, suggesting that contamination was responsible for the initial effect. An endotoxin concentration of less than 1.5 EU/mL, as observed in hB2M, could not induce oxidative stress.
Conclusion:
This study suggests that B2M, a traditional marker for middle molecule retention and a novel marker for cardiovascular outcome, may not by itself cause vascular damage by influencing inflammatory response due to induction of leukocyte free radical production. However, an effect on other cell types involved cannot be excluded. Our data further reveal that this type of research might be skewed by non-LPS contaminants, and that care should be taken to exclude this bias.
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