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First published online August 23, 2007
Stem Cells Vol. 25 No. 12 December 2007, pp. 3093 -3100
doi:10.1634/stemcells.2007-0525; www.StemCells.com
© 2007 AlphaMed Press

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THE STEM CELL NICHE

A Novel Role of Complement in Mobilization: Immunodeficient Mice Are Poor Granulocyte-Colony Stimulating Factor Mobilizers Because They Lack Complement-Activating Immunoglobulins

Ryan Recaa, Daniel Cramerb, Jun Yanb, Mary J. Laughlinc, Anna Janowska-Wieczorekd, Janina Ratajczaka, Mariusz Z. Ratajczaka,e

aStem Cell Institute at the James Graham Brown Cancer Center,
bTumor Immunobiology Program, University of Louisville, Louisville, Kentucky, USA;
cCase Western University, Cleveland, Ohio, USA;
dDepartment of Medicine, University of Alberta and Canadian Blood Services, Edmonton, Alberta, Canada;
eDepartment of Physiopathology, Pomeranian Medical University, Szczecin, Poland

Key Words. Complement • C3 • C5 • Mobilization • Granulocyte-colony stimulating factor • Zymosan • CXCR4

Correspondence: Mariusz Z. Ratajczak, M.D., Ph.D., Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202, USA. Telephone: (502) 852 1788; Fax: (502) 852 3032; e-mail: mzrata01{at}louisville.edu

Received July 9, 2007; accepted for publication August 14, 2007.
First published online in STEM CELLS EXPRESS   August 23, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Complement (C) and innate immunity emerge as important and underappreciated modulators of mobilization of hematopoietic stem/progenitor cells (HSPC). We reported that (a) C becomes activated in bone marrow (BM) during granulocyte–colony-stimulating factor (G-CSF)-induced mobilization by the classic immunoglobulin (Ig)-dependent pathway and that (b) C3 cleavage fragments increase the responsiveness of HSPC to a stromal derived factor-1 gradient. Since patients suffering from severe combined immunodeficiency (SCID) mobilize poorly, we hypothesized that this could be directly linked to the lack of C activating Ig in these patients. In the current study to better elucidate the role of C activation in HSPC mobilization, we mobilized mice that lack Ig (RAG2, SCID, and Jh) by G-CSF or zymosan, compounds that activate C by the classic Ig-dependent and the alternative Ig-independent pathways, respectively. In addition, we evaluated mobilization in C5-deficient animals. Mobilization was evaluated by measuring the number of colony-forming unit-granulocyte macrophage and leukocytes circulating in peripheral blood. We found that (a) G-CSF- but not zymosan-induced mobilization was severely reduced in RAG2, SCID, and Jh mice; (b) impaired G-CSF-induced mobilization was restored after infusion of purified wild-type Ig; and (c) mobilization was severely reduced in C5-deficient mice. These data provide strong evidence that the C system plays a pivotal role in mobilization of HSPC and that egress of HSPC from BM occurs as part of an immune response.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The forced migration of hematopoietic stem/progenitor cells (HSPC) from bone marrow (BM) into peripheral blood (PB) is called mobilization. Mobilization is important from a clinical point of view as a procedure that allows for the collection of HSPC for transplantation [1]. The molecular mechanisms governing mobilization of HSPC are still not well understood. However, accumulating evidence suggests that attenuation of the {alpha}-chemokine stromal derived factor-1 (SDF-1)-CXCR4 axis that plays a pivotal role in retention of HSPC in BM results in the release of these cells from the BM into PB [2–5].

Many agents have been described that induce mobilization of HSPC [5–8]. Granulocyte-colony stimulating factor (G-CSF), most frequently employed in the clinic, efficiently mobilizes HSPC after a few consecutive daily injections [9, 10]. Other compounds, such as polysaccharides (e.g., zymosan), mobilize HSPC within 1 hour after a single injection [11, 12]. Mobilization could also be induced by chemokines (e.g., IL-8, Gro-β), growth factors (e.g., vascular endothelial growth factor), and CXCR4 antagonists (e.g., AMD3100), and it is modulated by lipopolysaccharide that is released by intestinal bacteria [13–18]. Unfortunately, ~25% of patients do not respond efficiently to currently recommended mobilization protocols and are termed poor mobilizers.

Interestingly, it has been reported that patients suffering from severe combined immunodeficiency (SCID), who lack functional B and T lymphocytes, also mobilize poorly, which suggests that the immune system is involved in mobilization [19]. Lending support to this is our recent observation that innate immunity and cleavage fragments of the third component of the complement system (C3) play an important role in mobilization of HSPC [17]. We found that both C3- and C3aR-deficient mice are hypersensitive to G-CSF-induced mobilization and that a small molecular antagonist of C3aR, SB290157, enhanced G-CSF-induced mobilization in wild-type (wt) animals. This implies that bioactive C3 cleavage fragments (C3a and C3adesArg) play a role in retention of HSPC in BM. These data, combined with the observation that both C3a and C3adesArg enhance responsiveness of HSPC to an SDF-1 gradient, have established a new paradigm for the role of complement (C) in the balance between retention and mobilization of HSPC within the BM environment [2, 20–22].

Generally, C, which has emerged as a new modulator of HSPC trafficking, may be activated by the immunoglobulin (Ig)-dependent classic and Ig-independent alternative pathways. In a previous study, we reported that C is activated in the BM environment during mobilization with G-CSF, and the BM concentration of C3 cleavage fragments (C3a and C3adesArg) increases during such mobilization [17]. To explain this phenomenon, we postulate that G-CSF-induced mobilization, which turns BM into a highly proteolytic microenvironment [23], exposes a neoepitope in BM tissue [2, 24]. The newly exposed neoepitope then becomes recognized by naturally occurring circulating antibodies [24, 25], which activate C via the Ig-dependent classic pathway. In addition, C may also be activated in BM by the Ig-independent alternative pathway as seen, for example, after administration of zymosan [26].

In the current study, to better elucidate the role of C activation in triggering mobilization of HSPC, we performed mobilization studies in immunodeficient mice, RAG2, SCID, and Jh, which lack Ig and thus do not activate C through the classic pathway, and in their wt (control) littermates. We employed G-CSF to activate C by the Ig-dependent classic pathway and zymosan to activate it by the Ig-independent alternative pathway. We also performed mobilization in C5-deficient animals. Mobilization was evaluated by measuring the number of colony-forming unit-granulocyte macrophage (CFU-GM) and leukocytes circulating in peripheral blood. Our data support the concept that the C system is a major player in the egress of CFU-GM from BM into PB. Thus, in light of our findings, mobilization of HSPC could be envisioned as part of an immune response that requires C activation by the classic Ig-dependent (e.g., G-CSF) and/or Ig-independent (e.g., zymosan) pathways. Furthermore, we propose that different cleavage fragments of the activated C cascade affect stem cell mobilization in a negative (C3) or positive (C5) way.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mice
Pathogen-free, 4- to 6-week-old female C57BL/6, BALB/c, and BALB/c-SCID mice were purchased from the National Cancer Institute (Frederick, MD, http://www.cancer.gov). C57BL/6-RAG2 and BALB/c-Jh knockout mice were purchased from Taconic (Germantown, NY, http://www.taconic.com) and C57BL/6-C3 deficient mice (breeding colony) and C57BL/6-C5 deficient from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). Immunodeficient and C3-deficient animals were phenotyped (using radial immunodiffusion) for serum Ig or C3, respectively, prior to each experiment. Animal studies were approved by the Animal Care and Use Committee of the University of Louisville (Louisville, KY).

G-CSF Mobilization
Mice were injected subcutaneously with either 50 or 250 µg/kg of human G-CSF (Amgen, Thousand Oaks, CA, http://www.amgen.com) daily for 3 or 6 days. At 6 hours after the last G-CSF injection, mice were bled from the retro-orbital plexus to obtain white blood cell (WBC) counts and 450 µl of PB was obtained from the vena cava (with a 25-gauge needle and 1-ml syringe containing 50 µl of 100 mM EDTA). PB was spun down, plasma was collected, and pellets were lysed 2x in 10 ml of 1x BD Pharm Lyse buffer (BD Biosciences, San Jose, CA. http://www.bdbiosciences.com). WBC were subsequently washed twice and used in colony assays as described below.

Zymosan Mobilization
Mice were injected i.v. with 0.5 mg of zymosan. One hour after injection, the mice were bled from the retro-orbital plexus to obtain WBC counts, and 450 µl of PB was obtained from the vena cava (with a 25-gauge needle and 1-ml syringe containing 50 µl of 100 mM EDTA). PB was spun down, plasma was collected, and pellets were lysed 2x in 10 ml of lysis buffer (BD Biosciences). WBC were subsequently washed twice and used in colony assays as described below.

T-Cell Depletion
BALB/c mice were injected i.p. with 50 µg of rat anti-mouse CD4 (clone GK 1.5) and rat anti-mouse CD8 (clone 53-6.72) monoclonal antibodies (mAbs) on day –2 and day 3 of the 6-day G-CSF mobilization protocol. The antibodies were a gift of Nejat K. Egilmez of the University of Louisville. Depletion was analyzed by fluorescence-activated cell sorting (FACS) on PB of wt and CD4–CD8-depleted mice. A CD3 mAb (clone 17A2; BD Biosciences) was used for the FACS analysis.

Immunoglobulin Reconstitution
Mice were injected i.p. with 3 mg of purified mouse immunoglobulin (Equitech Bio Inc., Kerrville, TX, http://www.equitech-bio.com) from pooled mouse serum, mouse monoclonal IgG2a isotype control, 1.28 mg (a gift of Jun Yan of the University of Louisville), and mouse IgM, 0.52 mg (Bethyl Laboratories, Montgomery, TX, http://www.bethyl.com; MI10–100-6; with sodium azide dialyzed out), on days –1, 2, and 4 of the 6-day G-CSF mobilization protocol.

WBC Counts
Fifty microliters of PB was taken from the retro-orbital plexus of the mice and collected into microvette EDTA-coated tubes (Sarstedt Inc., Newton, NC, http://www.sarstedt.com/php/main.php). Samples were run within 2 hours of collection on a Hemavet 950 (Drew Scientific Inc., Oxford, CT, http://www.drew-scientific.com).

CFU-GM Colony Formation
We resuspended 2 x 105 WBC in 25% Iscove's modified Dulbecco's modified Eagle's medium plus 75% MethoCult H4230 (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 25 ng/ml recombinant murine granulocyte macrophage stimulating factor and 10 ng/ml recombinant murine interleukin-3 (StemCell Technologies). Cultures were incubated for 7 days, at which time they were scored for the number of granulocyte macrophage colonies under an inverted microscope.

Evaluation of Mobilization
The formula used for evaluation was (WBC [k/µl] x number of colonies)/number of WBC plated = number of CFU-GM per microliter of peripheral blood.

C3a Enzyme-Linked Immunosorbent Assay
After collection of blood as described above, samples were spun down at 300g for 10 minutes at 4°C. Serum was collected as the top fraction and either used immediately or frozen at –80°C. Briefly, a purified rat anti-mouse C3a antibody (Ab; clone I87-1162) was used as the capture Ab (2 µg/ml overnight at 4°C) and a biotin-labeled rat anti-mouse C3a Ab (clone I87-419) was used as the detection Ab (1 µg/ml, 1 hour at 37°C). Both Abs were purchased from BD Biosciences, and the recommended manufacturer's protocol was used to carry out the assay. Serum samples were used at a 1:10 dilution and incubated for 1 hour at 37°C. Purified mouse C3a (a gift from BD Biosciences) was used to generate the standard curve.

Deposition of iC3b
BM was flushed from the femurs of C57BL/6 or RAG2 mice with fresh medium. The cells were subsequently resuspended using an 18-gauge needle and filtered through a 70-µm nylon mesh and washed. The bone marrow cells were counted and resuspended in a single cell suspension at a concentration of 5 x 106 cells per milliliter. Two hundred microliters of the BM cell suspension was stained with 0.2 µl of affinity purified goat anti-mouse C3-Oregon Green (Immunology Consultant Laboratory Inc., Newberg, OR, http://www.icllab.com) and anti-mouse CD45-PE (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml). The cells were incubated for 30 minutes at 4°C in the dark. The samples were then washed 2x in phosphate-buffered saline and analyzed by flow cytometry using a BD FACSCalibur.

Statistical Analysis
Arithmetic means and standard deviations were calculated using Instat 3.0 (GraphPad Software Inc., San Diego, http://www.graphpad.com). Statistical significance was defined as * p < .05 or ** p < .01. Data were analyzed using Student's t test for unpaired samples.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Ig-Deficient RAG2, SCID, and Jh Mice, but Not T-Cell-Depleted Mice, Respond Poorly to G-CSF-Induced Mobilization
To test the role of Ig and C activation in G-CSF-induced mobilization of HSPC, immunodeficient RAG2, SCID (lacking both B and T lymphocytes and Ig), and Jh mice (selectively Ig-deficient) were mobilized by G-CSF for 3 or 6 days (Fig. 1). First, we observed that all of these mice with an unaffected myeloid compartment and normal numbers of CFU-GM in BM (not shown) under normal steady-state conditions have a slightly lower number of circulating HSPC and a significantly lower number of WBC in their PB compared with their wt littermates. This decrease was related to a severe deficiency of circulating lymphocytes. More importantly, the mobilization response to G-CSF of the Ig-deficient mice was lower than in wt mice. Figure 1, left panel, shows that all three types of immunodeficient mice have a significant decrease in the numbers of circulating clonogenic CFU-GM in their PB at days 3 and 6 of mobilization as compared with genotypically matched wt mice. Similarly, all Ig-deficient animals display a lower number of mobilized PB WBC counts (Fig. 1, right panel).


Figure 1
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  		Figure 1. G-CSF-induced mobilization is impaired in Ig-deficient RAG2, SCID, and Jh mice. SCID (A), RAG2 (B), and Jh (C) mice, as well as age and sex-matched wild-type (wt) mice, were mobilized for 3 or 6 days with G-CSF (250 µg/kg s.c. per day) (n = 9 animals per group). Data are pooled from three independent experiments. Left, number of circulating CFU-GM progenitors per microliter of peripheral blood; right, white blood cells (k/µl); * p ≤ .05, ** p ≤ .01 as compared with wt mice. Abbreviations: C, complement; CFU-GM, colony-forming unit-granulocyte macrophage; G-CSF, granulocyte–colony-stimulating factor; Ig Def., immunoglobulin deficient; SCID, severe combined immunodeficiency.

 
To exclude the possibility that T lymphocytes could account in part for the defective mobilization of RAG2 and SCID mice, we performed mobilization in T-cell-depleted mice. To achieve this, wt BALB/c mice were depleted of T lymphocytes using a cocktail of antibodies against CD4+ and CD8+ T lymphocytes. The efficiency of T depletion (98% ± 1.2% 2 days after administration of Abs) was confirmed by FACS analysis (not shown). Subsequently, wt control mice and T-cell-depleted mice were mobilized for 6 days by G-CSF (Fig. 2). We did not find any effect of T-cell depletion on the number of circulating CFU-GM in PB (Fig. 2, left panel), although the WBC count in PB was slightly reduced due to Ab-mediated T-cell depletion (Fig. 2, right panel).


Figure 2
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  		Figure 2. Depletion of T lymphocytes does not affect G-CSF-induced mobilization. BALB/c mice or BALB/c mice depleted of CD4+ and CD8+ cells were mobilized for 6 days with G-CSF (250 µg/kg s.c. per day) (n = 5 animals per group). Data are pooled from three independent experiments. Left panel: number of circulating CFU-GM progenitors per microliter of peripheral blood. Right panel: white blood cells (k/µl). Abbreviations: C, complement; CFU-GM, colony-forming unit-granulocyte macrophage; dep., depletion; G-CSF, granulocyte–colony-stimulating factor.

 
Defective Mobilization in RAG2, SCID, and Jh Mice Is Restored by Purified Immunoglobulins
To obtain proof that poor mobilization in RAG2, SCID, and Jh mice could be explained by a lack of C-activating Ig, we reconstituted these mice with purified wt Ig and, subsequently, mobilized them for 6 consecutive days with a suboptimal dose of G-CSF (50 µg/kg per day). The defective G-CSF mobilization observed in these mice was significantly improved if they were supplemented with wt Ig prior to G-CSF mobilization (Fig. 3, left panel) and corresponded with elevated PB WBC counts (Fig. 3, right panel). Of interest, Ig infusion enhanced mobilization not only in Ig-deficient but also in wt animals, particularly in C57BL/6 mice, which are regarded as poor mobilizers [27] (Fig. 3B); however, this effect was less pronounced when mobilization was forced by a high dose of G-CSF (250 µg/kg per day). This indicates that wt Ig contains an admixture of naturally occurring antibodies that are able to recognize a neoepitope and activate C in BM that has been exposed to G-CSF [25, 28]. The requirement for wt Ig was subsequently supported in the experiments in which irrelevant purified monoclonal IgG or IgM antibodies were used and no increase in mobilization was observed (Fig. 4). The potential contamination of Ig preparations by endotoxin, which alone can mobilize HSPC [29], was excluded by showing enhancing effect of Ig plus suboptimal dose of G-CSF in C3He/J mice (data not shown). In addition, Ig preparations alone did not mobilize mice.


Figure 3
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  		Figure 3. G-CSF-induced mobilization is restored after supplementation with Ig. SCID (A), RAG2 (B), and Jh (C) mice, as well as age- and sex-matched wild-type mice, not supplemented or supplemented with purified mouse immunoglobulin were mobilized for 6 days with G-CSF (50 µg/kg s.c. per day) (n = 7 animals per group). Data are pooled from three independent experiments. Left: number of circulating CFU-GM progenitors per microliter of peripheral blood; * p ≤ .05, ** p ≤ .01 as compared with 6-day G-CSF-mobilized mice. Right: white blood cells (k/µl); * p ≤ .05, ** p ≤ .01 as compared with wild-type mice. Abbreviations: C, complement; CFU-GM, colony-forming unit-granulocyte macrophage; G-CSF, granulocyte–colony-stimulating factor; Ig, immunoglobulin; Ig Def., immunoglobulin deficient; SCID, severe combined immunodeficiency.

 


Figure 4
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  		Figure 4. Irrelevant monoclonal immunoglobulins are not sufficient to enhance G-CSF-induced mobilization. RAG2 mice were treated with either purified mouse immunoglobulin, monoclonal isotype control IgG, or monoclonal isotype control IgM and subsequently mobilized with G-CSF (50 µg/kg s.c. per day) (n = 4 animals per group). Data are pooled from two independent experiments. Number of circulating CFU-GM progenitors per microliter of peripheral blood; ** p < .01 as compared with 6-day G-CSF mobilized mice. Abbreviations: CFU-GM, colony-forming unit-granulocyte macrophage; G-CSF, granulocyte–colony-stimulating factor; Ig, immunoglobulin; wt, wild type.

 
Complement Activation and C3a Generation in Serum Correlate with G-CSF-Induced HSPC Mobilization
We reported that, in G-CSF-induced mobilization, C3 was cleaved and C3adesArg (liquid phase C3 cleavage product) became detectable by enzyme-linked immunosorbent assay in serum [17]. At the same time, the solid phase C3 cleavage product, iC3b, was deposited on BM-derived fibroblasts and endothelial cells as determined by FACS analysis of these cells stained with anti-iC3b antibodies [20].

To test directly whether C becomes activated during G-CSF mobilization, we first tested the serum of wt, RAG2, SCID, and Jh mice for the presence of C3a (soluble C3 cleavage fragment) before and after G-CSF mobilization. The serum of G-CSF-mobilized animals revealed impaired C activation/cleavage in Ig-deficient animals and its increase in animals supplemented with wt Ig (Fig. 5A). As expected, no C activation/cleavage was detected in C3–/– mice (data not shown).


Figure 5
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  		Figure 5. Complement activation products C3a and iC3b are detectable following mobilization. (A): C3a fold change detected by sandwich enzyme-linked immunosorbent assay (ELISA) in serum of G-CSF (250 µg/kg s.c. per day) mobilized BALB/c, SCID, Jh, C57Bl/6, and RAG2 mice with or without supplementation with purified mouse immunoglobulin (n = 6 animals per group); * p ≤ .05, ** p ≤ .01 as compared with 6-day G-CSF mobilized mice. (B): C3a fold change detected by sandwich ELISA in serum of zymosan-mobilized BALB/c, SCID, Jh, C57Bl/6, and RAG2 mice (n = 6 animals per group); * p ≤ .05, ** p ≤ .01 as compared with unmobilized mice. (C): Bone marrow mononuclear cells (BM MNC) were flushed from the femurs of RAG2 or C57BL/6 mice and subsequently stained with an anti-mouse iC3b antibody. The increase in mean fluorescent intensity as analyzed by fluorescence-activated cell sorting over BM MNC from unmobilized mice is shown. Data are pooled from two independent experiments; * p < .05 as compared with unmobilized mice. Abbreviations: G-CSF, granulocyte–colony-stimulating factor; Ig, immunoglobulin; MFI, mean fluorescent intensity; SCID, severe combined immunodeficiency.

 
Next, using FACS, we evaluated iC3b deposition on BM mononuclear cells (MNC) in Ig-deficient RAG2 mice mobilized by G-CSF. Figure 5C shows no increase in iC3b deposits in RAG2 animals mobilized by G-CSF compared with wt C57BL/6 mice, although an increase is detectable on BM MNC if the mice are supplemented with Ig during mobilization.

RAG2, SCID, and Jh Mice Display Normal Zymosan-Induced Mobilization
Zymosan activates C by the Ig-independent alternative pathway. To better understand the role of Ig and the classic pathway of C activation in mobilization, we performed similar mobilization studies in RAG2, SCID, and Jh mice employing zymosan as a mobilizing agent. These studies revealed that these immunodeficient animals respond to zymosan like their wt-matched controls (Fig. 6), and mobilization of CFU-GM was again associated with C activation/cleavage (Fig. 5B). Notably, C3–/– mice did not mobilize CFU-GM in response to zymosan (data not shown).


Figure 6
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  		Figure 6. Zymosan-induced mobilization is normal in immunoglobulin-deficient SCID, Jh, and RAG2 mice. (A): Jh, SCID, and BALB/c mice were mobilized for 1 hour with zymosan (0.5 mg i.v.) (n = 9 animals per group). (B): RAG2 and C57BL/6 mice were mobilized for 1 hour with zymosan (0.5 mg/kg i.v.) (n = 9 animals per group). Data are pooled from three independent experiments. Left: number of circulating CFU-GM progenitors per microliter of peripheral blood. Right: white blood cells (k/µl); * p ≤ .05, ** p ≤ .01 as compared with wild-type mice. Abbreviations: CFU-GM, colony-forming unit-granulocyte macrophage; Ig Def., immunoglobulin deficient; SCID, severe combined immunodeficiency.

 
These results further support the evidence for a pivotal role of C activation in the mobilization of HSPC. Since zymosan activates C by the alternative pathway, all Ig-deficient (RAG2, SCID, and Jh) mice mobilize normally.

Impaired Mobilization in C5-Deficient Mice Supports a Pivotal Role for C in Mobilization of HSPC
In a previous study, we reported that C3 cleavage fragments increase responsiveness of HSPC to an SDF-1 gradient and postulated that C3a and C3adesArg provide a "last line of defense" to protect HSPC from uncontrolled egress from BM during mobilization [17]. The current data, however, show that activation of the C cascade downstream from C3 is crucial to execute both G-CSF- and zymosan-induced egress of HSPC.

Since both classic and alternative pathways of C activation merge at C3, subsequently leading to the activation of C5, to better address the role of C in mobilization, we also performed mobilization studies in C5-deficient mice. We found that mobilization in C5 deficient mice was severely suppressed in response to both G-CSF (Fig. 7A) and zymosan (Fig. 7B). This observation further confirms the role of the C cascade as a pivotal modulator of stem cell mobilization. Furthermore, the different effect of C3 and C5 cleavage fragments on mobilization of HSPC suggests that the C cascade modulates egress of HSPC in both a negative (C3) and a positive (C5) manner (Fig. 7C).


Figure 7
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  		Figure 7. Impaired mobilization in C5 deficient mice. C5 deficient as well as age- and sex-matched C5 sufficient mice were mobilized (A) for 6 days with G-CSF (250 µg/kg s.c. per day) (n = 6 animals per group) or (B) for 1 hour with zymosan (0.5 mg/kg i.v.) (n = 6 animals/group). Data are pooled from three independent experiments. Left: number of circulating CFU-GM progenitors per microliter of peripheral blood. Right: white blood cells (k/µL); * p ≤ .05, ** p ≤ .01 as compared with C5 sufficient mice. (C): Complement cascade activation products differentially modulate stem cell trafficking. Upon activation of the C system, C3 and C5 cleavage products play opposite roles in the retention or mobilization of cells, respectively. While liquid phase C3 cleavage fragments (C3a and C3adesArg) enhance responsiveness of hematopoietic stem/progenitor cells (HSPC) to a stromal derived factor-1 gradient, solid phase cleavage fragment iC3b deposited onto surrounding surfaces helps to retain HSPC in their niche. In contrast, as we hypothesize, activation of C5 promotes mobilization of HSPC by increasing the permeability of the bone marrow endothelium and the recruitment/degranulation of granulocytes. Thus, we propose that an activated C cascade can affect stem cell mobilization in a negative (C3) or a positive (C5) way. The potential contribution of other C cascade proteins (e.g., MAC) requires further study. Abbreviations: C, complement; CFU-GM, colony-forming unit-granulocyte macrophage; G-CSF, granulocyte–colony-stimulating factor; Ig, immunoglobulin; MAC, membrane attack complex.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
We postulated that innate immunity and the C system modulate mobilization of HSPC, which could be envisioned as an element of the immune response [2, 30]. Since patients suffering from severe combined immunodeficiency mobilize poorly with G-CSF [19] and C becomes activated/cleaved in BM after G-CSF administration via the classic Ig-dependent pathway [17, 25, 31], we hypothesized that this defect observed in SCID patients could be directly linked to the lack of C activating Ig. Thus, to better elucidate the role of Ig in C activation and triggering mobilization of HSPC, we performed mobilization studies in various strains of immunodeficient mice that lack Ig and thus cannot activate C by the classic Ig-dependent pathway. We also employed zymosan, which activates C through the alternative Ig-independent pathway. Finally, since both classic and alternative C activation pathways merge at the level of C3 [32, 33], and activation of C3 subsequently leads to C5 cleavage, mobilization studies were also performed in C5-deficient mice.

We report here for the first time that immunodeficient mice lacking Ig, in contrast to their genetically matched wt counterparts, respond poorly to G-CSF-induced mobilization. At the same time, however, they respond normally to zymosan.

The model of G-CSF-induced HSPC mobilization is the one best studied so far, and evidence from several laboratories suggests that attenuation of the SDF-1–CXCR4 axis precedes egress of HSPC from the BM into PB [2–5]. It is proposed that, after G-CSF infusion, the BM turns into a proteolytic microenvironment that leads to enzymatic degradation of SDF-1 and enzymatic processing of the N-terminus of CXCR4 [23]. In the process of proteolysis of SDF-1 and CXCR4, various proteolytic enzymes are secreted by myeloid precursors and granulocytes in BM, such as elastase, cathepsin, and metalloproteinases [34, 35]. Furthermore, as recently reported, G-CSF may directly downregulate the expression of SDF-1 at the mRNA level in BM [36]. The end result is a decrease in responsiveness of HSPC to an SDF-1 gradient and their release into circulation.

Based on our previous [17] and current data, we propose that G-CSF-induced proteolytic processes in the BM also lead to "BM injury," which triggers the local activation of C. The mechanism of C3 activation involves mostly the classic (Ig-dependent) but also, to some degree, the alternative (Ig-independent) C activation pathways [32, 33, 37]. The former is triggered after exposure of a neoepitope in BM tissue damaged by secreted proteases. The neoepitope binds naturally occurring antibodies that circulate in peripheral blood and via C1 (q) triggers C activation through the classic pathway [24, 28]. Furthermore, it is likely that C3 could also be cleaved/activated by G-CSF-induced proteolytic proteases [34, 38]. Similarly, since G-CSF activates the coagulation system [39–41] and thrombin has recently been identified as a C5 activator [37], C could also be activated as a result of activation of the coagulation cascade.

Thus, several pathways could potentially be involved in C activation after G-CSF administration. However, by employing Ig-deficient mouse models, we report evidence that the classic Ig-dependent pathway of C activation is crucial for optimal G-CSF-induced mobilization. Supporting this notion are the facts that all Ig-deficient mice mobilized poorly with G-CSF and supplementation with wt Ig increased their mobilization efficiency. Interestingly, Ig also increased mobilization in wt animals, in particular C57BL/6 mice, which are known as a poorly mobilizing strain [42]. However, since some degree of mobilization occurred in Ig-deficient mice, C must become activated during G-CSF administration by alternative mechanisms as well. In fact, C3a cleavage fragments remained detectable in the serum of these animals. Furthermore, we also demonstrate here that zymosan-induced mobilization is not altered in Ig-deficient mice, which is to be expected, as this compound activates C by the Ig-independent alternative pathway [26]. Thus, evidence from both types of mobilization suggests that C activation and C3 cleavage are prerequisites for mobilization to occur.

Our recent observations that C5-deficient and Ig-deficient mice, which do not activate C by the classic pathway, are poor mobilizers support this notion. However, this conclusion does not, at first glance, corroborate our previous report on the mobilization of C3–/– and C3aR–/– mice, which we reported to be easy mobilizers [17]. This apparent discrepancy can be explained by the fact that the various C cleavage fragments have different effects on mobilization. Based on the previous and current data, a more complex picture of the role of C in HSPC mobilization has now emerged.

We hypothesize that mobilization/retention of HSPC in BM is regulated differentially at various levels of the C activation cascade (Fig. 7C) and that the C system may contain internal checks and balances that modulate trafficking of HSPC. We believe that the soluble (C3a, C3adesArg) and solid (iC3b) phases of C3 cleavage are primarily involved in retention of HSPC in BM. In this context, the BM microenvironment, by expressing C3a and C3adesArg, is increasing the responsiveness of HSPC to an SDF-1 gradient as a last line of defense against an uncontrolled egress of HSPC from BM into PB [2, 17, 20]. Similarly, iC3b deposited in the BM microenvironment tethers HSPC and increases their retention in BM [20, 24]. This explains why C3- or C3aR-deficient mice that lack this last line of defense are easy mobilizers.

On the other hand, C3 activation is obligatory for activation of downstream C proteins including C5, and we demonstrate here that lack of C5 activation is associated with impaired mobilization of HSPC (Fig. 7A, 7B). An explanation of this phenomenon might be that C5 cleavage fragments (C5a and C5adesArg) are potent anaphylatoxins that increase the vascular permeability of BM vasculature as well as activate granulocytes to release proteolytic enzymes [32, 33, 43], which could potentially activate C by alternative mechanisms.

Overall, our data support the idea that C is involved in crucial mechanisms responsible for the egress of HSPC from the BM. On the other hand, since the generation of two less potent anaphylatoxins (C3a and C4a) is not impaired in C5-deficient mice, this explains why some HSPC are still mobilized even in the total absence of C5a. Furthermore, it is also likely that some other downstream C proteins (e.g., C6 or the membrane attack complex) may be required for the egress of HSPC from BM. This issue will be addressed by performing mobilization in C6-deficient mice. Similarly, selective depletion of neutrophils in normal mice will address involvement of peripheral blood mononuclear cells on C-mediated mobilization. Finally, competitive repopulating studies using mobilized peripheral blood mononuclear cells will address the effect of C on mobilization of the most primitive HSC. It will be also important to evaluate a number of mobilized most primitive Sca-1+kit+lin cells that do not express CD34 antigen [44]. We are currently testing these possibilities in our laboratory.

In conclusion, our data support the concept that the C system is a major factor modulating egress of CFU-GM from BM into PB. Thus, in light of our findings, mobilization of HSPC could be envisioned as part of an immune response that requires C activation by the classic Ig-dependent and/or Ig-independent pathways. Hence, modulation of C activation could allow for the development of more efficient mobilization strategies in patients who are poor mobilizers of HSPC.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by NIH Grant R01 DK074720-01 and KBN grant 2POSA-00429 to M.Z.R.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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