Complement cascade (CC) and innate immunity have emerged as important modulators of hematopoietic stem/progenitor cell (HSPC) trafficking. We reported that the CC becomes activated in bone marrow (BM) during HSPC mobilization induced by G-CSF or AMD3100 and proposed that the generation of C5a and C5b-C9 (membrane attack complex; MAC) is required for optimal mobilization (Stem Cells 2007; 25:3093; Leukemia 2010; 24:976). While C5a induces a proteolytic microenvironment in BM that attenuates SDF-1-CXCR4 retention signals for HSPC and promotes egress of leucocytes, C5b-C9 (MAC) induces the release of a crucial chemoattractant, sphingosine-1 phosphate (S1P), from red blood cells and augments mobilization of HSPC from BM. On the other hand, C3 cleavage fragments attenuate mobilization by enhancing responsiveness of HSPC to SDF-1 retention signals thus promoting HSPC retention in BM. Thus our findings suggest that mobilization is differently regulated by the proximal and distal parts of CC (upstream and downstream of C3, respectively). We also demonstrated that C3-deficient mice (lacking C3 cleavage fragments) are easy mobilizers whereas C5-deficient mice (lacking C5 cleavage fragments and not generating MAC) mobilize HSPC very poorly (Leukemia 2009; 23:2052). C4 is part of the classical pathway of CC whose activation/cleavage is initiated by C1 and C2, releasing smaller (C4a and C2b) and larger (C4b and C2a) fragments. C2a binds with C4b to form an enzymatic complex termed C3 convertase that cleaves C3 into C3a anaphylatoxin and C3b.
In the present study we investigated the role of C4 in HSPC mobilization and hypothesized that C4-deficient mice are easy mobilizers, supporting the notion that the proximal part of CC is crucial for retention of HSPC in the BM microenvironment.
We employed 6–8 week old C4b-deficient mice (strain B6.129S-C4btm1Crr/J from Jackson Laboratory, Bar Harbour, ME) and wild type (WT) littermates. Mice were injected subcutaneously with 250 μg/kg of human recombinant G-CSF (Amgen, Thousand Oaks, CA) or saline (control) daily for 3 days. At 6 h after the last G-CSF injection, the mice were sacrificed and blood was collected from the vena cavae. Mobilization was evaluated by determining the number of leukocytes (WBC) and colony-forming unit granulocyte-macrophages (CFU-GM) circulating in the peripheral blood (PB). We also measured the level of mouse terminal complement complex C5b-C9 (MAC) in plasma using ELISA (Kamiya) in G-CSF-mobilized and non-mobilized C4b-deficient and WT mice.
We found that C4b-deficient mice treated with saline have slightly higher WBC counts than WT mice and that G-CSF induced a greater increase in WBC counts in these mice than in WT mice. Based on CFU-GM counts after G-CSF mobilization, C4b-deficient mice mobilized significantly more HSPCs into PB than WT mice (p < 0.0065). These observations were consistent in all four experiments performed involving 18–20 mice per experiment. The G-CSF mobilization responses correlated with a greater increase in activation of the distal part of CC in C4b-deficient mice compared to WT mice, as evidenced by C5b-C9 levels evaluated by ELISA.
Our data indicate that C4-deficient mice are easy mobilizers, thus supporting a role for the proximal part of CC in retention of HSPC in BM. Furthermore, mobilization of HSPC in C4b-deficient mice correlated with activation of the distal part of CC and generation of C5b-C9 (MAC) suggesting that activation of the distal part of CC is crucial for egress of HSPCs into PB. Moreover, activation of the distal part of CC in C4-deficient mice that have defective activation of the proximal part of CC indicates that C5 may be cleaved in a C3-independent manner by other proteolytic enzymes present in blood plasma. Finally, our data further support that mobilization is part of a more general immune response in which all complement components, including C4, play a significant role.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.