G-CSF induces leukocyte recruitment in vivo via the leukocyte G-CSF receptor
JL Eyles, IK Campbell, IP Wicks in collaboration with BA Croker, A Roberts, D Metcalf (Cancer and Haematology Division), MJ Hickey, MU Norman (Centre for Inflammatory Diseases, Monash University, Clayton)
Granulocyte Colony-Stimulating Factor (G-CSF) is in routine clinical use, but can activate neutrophil adhesion in vitro. The aim of this study was to determine if G-CSF is capable of inducing alterations in leukocyte-endothelial cell interactions in vivo. Mice were treated with G-CSF either by acute, local administration, or systemically over four hours, and leukocyte-endothelial cell interactions assessed via intravital microscopy of the cremasteric microcirculation. Local application of G-CSF caused a reduction in leukocyte rolling, but a significant increase in leukocyte adhesion within 15 minutes, a process dependent on CD11b (Mac-1). In vivo confocal microscopy revealed that the adherent cells were exclusively Gr-1+ neutrophils. Adherent leukocytes also underwent transmigration within 45 minutes. To assess the effect of G-CSF under conditions mimicking its clinical use, G-CSF was also administered systemically. Systemic G-CSF induced increased leukocyte rolling, adhesion and transmigration after four hours. Using mice rendered chimeric for the G-CSF receptor (G-CSFR) via bone marrow transfer between wild-type and Gcsfr-/- mice, this response was found to be dependent on leukocyte-expressed G-CSFR. These findings indicate a pro-inflammatory role for G-CSF and provide a potential mechanism for the association of G-CSF administration with the exacerbation of diseases such as rheumatoid arthritis.
Acute local administration of G-CSF promotes Mac-1-dependent adhesion and transendothelial migration of neutrophils in vivo. (A) Leukocyte rolling and (B) adhesion in postcapillary venules (vessel diameter 25-40 µm) of cremaster muscle before and after superfusion with saline or 15 µg/ml G-CSF for up to 30 minutes in wild-type mice, *P < 0.05, **P < 0.001 versus basal adhesion, or versus saline rolling flux. (C) In vivo confocal image using PE-conjugated anti-Gr-1 mAb showing that adherent cells in G-CSF-treated muscle are all Gr-1+ (neutrophils). (D) Leukocyte rolling, (E) adhesion, and (F) transendothelial migration in postcapillary venules of cremaster muscle before and after with 15 µg/ml G-CSF for up to 45 minutes in wild-type mice that were pre-treated with either anti-Mac-1 mAb (clone 5C6) or an isotype control mAb treated mice. Data represent mean ± s.e.m.*P < 0.05, **P < 0.001
Synovial macrophages promote the local differentiation of Th17 cells during acute inflammatory arthritis
AE van Nieuwenhuijze, PJ Egan, IK Campbell, IP Wicks
The mBSA/IL-1 model of acute inflammatory arthritis is dependent on IL-17 production by Th17 cells. We found that synovial macrophages taken from inflamed joints support Th17 differentiation of naïve CD4+ T cells in co-culture experiments, but macrophages taken from the peritoneum of arthritic mice do not. This effect is dependent on cell-cell contact and can be inhibited by a blockade of IL-6. These results suggest that the macrophages in the inflamed joint might be intrinsically different from those in other tissues – synovial macrophages produce more IL-6, express higher levels of IL-1β, TGFβ and IL-23p19, and thereby promote the local differentiation of pro-inflammatory Th17 cells.