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Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. However, the function of neutrophils in MM and monoclonal gammopathy of undetermined significance (MGUS) has been poorly investigated.

The objective of this investigation was evaluating the activation status of peripheral neutrophils (N) isolated from MM and MGUS patients

Using oligonucleotide microarrays we first evaluated the gene expression profile (GEP) of granulocytes at the steady state in 10 MM, 5 MGUS and 8 healthy subjects matched for sex and age, identifying Arg-1 and PROK-2 among the first 10 genes differentially expressed in MM versus healthy granulocytes.

Thus, we validated Arg-1 and PROK-2 by RT-PCR in a series of 60 newly-diagnosed MM patients, 30 MGUS and 30 healthy subjects.

Activation status on N was then investigated in the same series, evaluating phagocytic and burst activity by commercial kit, surface expression of CD64, CD16, CD62L and CD11b, markers of neutrophil activation and reactive oxygen species (ROS) by flow cytometer.

Finally, we tested the immunosuppressive properties of N isolated from MGUS or MM patients, through functional assays, based on in vitro co-culture of N isolated from patients and T-lymphocytes from healthy subjects. 

MM-N exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. PROK-2 expression was two times higher in MGUS than healthy subjects (p=.02) and up to ten times higher in MM (p=.001). In MM patients, increased levels of PROK-2 were positively associated with advanced bone disease and unfavourable cytogenetics.

The phagocytic activity was reduced in MM-N compared to HS-N and MGUS-N (46% versus 75% versus 72%, p<0.0001 and p<0.0001  respectively).

Oxidative burst was 71% in MM-N, lower than HS-N (85%, p=0.01) and MGUS-N (86%, p=0.01). The MM-N exhibit higher levels of ROS (MFI 113 ± 9) compared to HS-N (MFI 46 ± 7) and MGUS-N (MFI 62 ± 8, p<0.0001 and p=0.0001 respectively).

Expression of CD64 was significantly elevated in MM-N compared to MGUS-N or healthy subjects-N (p=0.01 and p= 0.007 respectively) and was inversely correlated with phagocytic activity (p=0,01).

After 72 hours, MM-N were able to reduce T-cell proliferation at both tested 1:2 and 1:8 ratios, while MGUS-N could induce a similar effect only at the highest ratio 1:8. This effect was partially reverted with the treatment of 200 μM nor-NOHA, an Arg-1 inhibitor.

Compared to controls, neutrophils obtained from MM patients have a reduced phagocytic activity, a greater expression of Arg-1 and PROK-2 and exhibit an immunosuppressive function on T lymphocytes. These features are only minimally present in neuthophils from MGUS patients