Pro‐Inflammatory Cytokines and the Marrow Microenvironment
Authors: Lee Seungyong , Prisby Rhonda 
Published: November 2023

In the presence of marrow, vasodilation of the femoral principal nutrient artery (PNA; the primary conduit for blood flow to long bone) was impaired in young (p<0.05; Prisby et al., 2014, ASBMR) and old (p=0.07; Lee et al., 2014, ASBMR) rats. We also reported bone marrow blood vessel ossification (i.e., the conversion of bone marrow blood vessels into bone) in rats (Prisby, 2014) and mice (Lee and Prisby, 2016, ASBMR). Since chronic exposure to marrow‐derived pro‐inflammatory cytokines was closely associated with atherosclerotic lesions (Erbel et al., 2014), we theorized that the marrow microenvironment contributes to both bone vascular dysfunction and bone marrow blood vessel ossification. Physiological saline solution (PSS) from previous experiments analyzing vasodilation of the femoral PNA in young (n=3) and old (n=3) rats in the presence (MARROW) and absence (CON) of marrow was collected, snap frozen and stored at −80°C. The PSS was subsequently analyzed for the presence of cytokines (i.e., EPO, G‐CSF, GM‐CSF, GRO/KC, IFN‐γ, IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12p70, IL‐13, IL‐17A, IL‐18, M‐CSF, MCP‐1, MIP‐1α, MIP‐3α, RANTES, TNF‐α and VEGF). Marrow mass was similar between groups (young; 72±21 mg vs. old 93±24 mg). Eleven of the cytokines (i.e., EPO, G‐CSF, GRO/KC, IFN‐γ, IL‐2, IL‐5, IL‐7, IL‐12p70, IL‐17A, IL‐18 and TNF‐α) were undetectable in the PSS and therefore not quantified. Four cytokines were not different among groups (i.e., IL‐13, MIP‐1α, MIP‐3α, and VEGF). Pro‐inflammatory cytokines IL‐4, IL‐1α and IL‐1β were increased (p<0.05) in the presence of marrow. IL‐4 was enhanced (p<0.05) in young MARROW (0.58±0.04 pg/ml) vs. the other groups (young CON, 0.31±0.06 pg/ml; old CON, 0.26±0.02 pg/ml and old MARROW, 0.32±0.03 pg/ml). Similarly, IL‐1α was higher (p<0.05) in young MARROW (3.5±0.7 pg/ml) vs. young CON (1.0±0.3 pg/ml) and old CON (0.7±0.3 pg/ml), and tended (p=0.053) to be higher than old MARROW (1.9±0.5 pg/ml). IL‐1β was increased (p<0.05) in young MARROW (16.3±1.4 pg/ml) and old MARROW (20.9±3.0 pg/ml) vs. young CON (3.0±0.3 pg/ml) and old CON (2.2±0.3 pg/ml), and IL‐1β tended (p=0.091) to be higher in old MARROW vs. young MARROW. Additionally, the pro‐inflammatory cytokine IL‐6 was higher (p<0.05) in young (CON, 4.2±0.1 pg/ml and MARROW, 4.7±0.7 pg/ml) compared to old (CON, 2.1±0.9 pg/ml and MARROW, 1.8±0.5 pg/ml). In the presence of marrow, MCP‐1 tended (p=0.053) to be higher than in the CON groups (young MARROW, 11.4±1.8 pg/ml and old MARROW, 11.4±1.3 pg/ml vs. young CON, 6.2±0.4 pg/ml and old CON, 6.3±0.6 pg/ml). RANTES was higher (p<0.05) in old MARROW (89.6±32.5 pg/ml) vs. young CON (15.0±0.9 pg/ml), young MARROW (20.0±0.6 pg/ml) and old CON (13.3±0.7 pg/ml). However, GM‐CSF tended (p=0.067) to be lower in young MARROW (0.5±0.2 pg/ml) and old MARROW (0.5±0.2 pg/ml) vs. young CON (1.2±0.1 pg/ml). Anti‐inflammatory cytokine, IL‐10, tended (p=0.055) to be higher in young MARROW (43.5±5.5 pg/ml) vs. young CON (27.0±1.1 pg/ml) and old CON (26.4±2.1 pg/ml). We conclude that pro‐inflammatory cytokine production was enhanced in PSS containing the marrow, which may contribute to vascular dysfunction and the ossification of bone marrow blood vessels.Support or Funding InformationNIH NIAMS 7R15AR062882‐02 and NIH NIGMS P20 GM103446 Core Access Award (R. Prisby)