ARTICLE |
CORRESPONDENCE Jean-Pierre Lévesque: jplevesque{at}mmri.mater.org.au OR Ingrid G. Winkler: iwinkler{at}mmri.mater.org.au
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hemopoietic progenitor cells (HPCs) are responsible for the renewal of all mature blood cells. In adult mammals, the majority of HPCs reside in the BM. Transient increases in the number of HPCs circulating in the peripheral blood (mobilization) occur in response to a wide variety of stimuli including strenuous physical exercise, myelosuppressive chemotherapy, polyanions, chemokines, and hematopoietic growth factors (1).
Mobilized HPCs are now the favored source of transplantable cells to reconstitute hematopoiesis after high-dose chemotherapy. Currently, the agent most commonly used to elicit HPCs mobilization is G-CSF used alone or in combination with myelosuppressive chemotherapy (2, 3). The administration of G-CSF induces a 10- to 100-fold increase in the level of circulating HPCs in both humans and mice. G-CSFinduced mobilization is time and dose dependent, involving a rapid neutrophilia (evident within hours) and a gradual increase in HPC numbers in the blood peaking between 4 and 7 d of G-CSF administration. Mobilization with chemotherapeutic agents such as cyclophosphamide (CY) occurs during the recovery phase after the chemotherapy-induced neutropenia, that is, days 68 in mice, and days 1014 in humans. Although mobilized HPCs collected from the peripheral blood are extensively used to rescue hematopoiesis in patients undergoing high-dose myeloablative chemotherapy, the exact molecular mechanisms responsible for the mobilization of HPCs from the BM into the peripheral blood remain unclear.
Essential for the retention of HPCs in the BM are adhesive and chemotactic interactions. Particularly important are (a) the adhesive interaction between the vascular cell adhesion molecule VCAM-1 (CD106) expressed by the BM stroma with its counter receptor integrin 4ß1 (VLA-4) expressed by HPCs, and (b) HPC chemotaxis due to binding of the chemokine CXCL12 (SDF-1) produced by the BM stroma, to its cognate receptor CXCR4 (CD184) expressed at the surface of HPCs. Blocking either of these interactions; the VCAM-1
4ß1 adhesive interaction (46) or the CXCL12CXCR4 chemotaxic interaction (7, 8), by means of antibodies, antagonists, or tissue-specific gene-targeted deletion has been shown to result in mobilization of HPCs in vivo.
Our group has shown that HPC mobilization induced by G-CSF or CY coincides with the accumulation of high concentrations of active neutrophil serine proteases within the BM (9, 10). The predominant proteases were identified as neutrophil elastase (NE) and cathepsin G (CG), both released by neutrophils upon activation (9, 10). Once released into the BM extracellular fluid, these proteases can disrupt locally the two important mechanisms by which HPCs home to and remain within the BM: (a) adhesion to the BM stroma by cleaving the extracellular domain of VCAM-1 (9, 10), and (b) chemotaxis of HPCs by degrading and inactivating the chemokine CXCL12 (11, 12) and cleaving the 1st extracellular domain of CXCR4 expressed by human HPCs (12). As previous studies have shown, inactivation of either is sufficient to induce mobilization. However, a mechanism explaining how high concentrations of active neutrophil serine proteases accumulate in the BM during mobilization remains unclear.
Neutrophil serine protease activity is controlled in vivo by naturally occurring serine protease inhibitors or serpins (13, 14). Serpins function like mousetraps with a protruding reactive center loop. Cleavage of this reactive center loop by a specific protease triggers a spring-like mechanism, which entraps the protease within the serpin structure destroying the protease catalytic architecture (1517). Once formed, the inactive serpinprotease complex is rapidly cleared from the body. Although serpin structure is very conserved, the reactive center loop sequence is highly variable between different serpins allowing the targeting of specific proteases. Two groups of serpins located on chromosome 14 in humans and chromosome 12 in mice (referred to as "clade A" serpins; 14, 18) can inactivate neutrophil serine proteases in blood and tissues. These are SERPINA1 (also known as 1-proteinase inhibitor or
1-antitrypsin: AAT) and SERPINA3 (
1-antichymotrypsin: ACT; 19, 20). Although humans express a single copy of each SERPINA1 and SERPINA3 gene, in the house mouse Mus musculus the serpina1 gene has replicated five times (serpina1ae; 2123) whereas the serpina3 gene has replicated 14 times (serpina3an; 23, 24).
To explain how high concentrations of active neutrophil serine proteases accumulate in the bone marrow during HPC mobilization, we hypothesize that (a) serine protease inhibitors, in particular serpins, are present in steady-state BM preventing the action of serine proteases released by BM neutrophils in steady-state conditions, and (b) that the balance between serine proteases and their inhibitors shifts in mobilized BM leading to the accumulation of active NE and CG.
Herein we report that, serpina1 and serpina3 are both transcribed in steady-state BM and are present at sufficient concentrations to inhibit local serine proteases. During mobilization induced by either G-CSF or CY, both protein and mRNA concentrations of serpina1 and serpina3 in the BM dramatically drop associated with an accumulation of active neutrophil serine proteases and concomitant cleavage and inactivation of molecules essential for the retention of HPCs within the BM.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Levels of serpina1 protein drop in the BM during mobilization
Serpina1 protein, the main inhibitor of NE in tissues, was analyzed in BM extracellular fluids by immunoblotting. Whereas serpina1 (53 kD) was present in BM fluids before and after mobilization, levels were significantly decreased during HPC mobilization on days 26 after G-CSF administration (Fig. 2 A). Similarly, levels of serpina1 were significantly decreased in the BM fluids from CY-treated mice between days 610 again corresponding to the peak of mobilization in these mice (Fig. 2 B). This decrease in serpina1 was restricted to the BM as no corresponding change in serpina1 was observed in the matching plasma from these same mice (Fig. 2, A and B, lower panels).
|
In another series of experiments, serpina1 concentration in BM fluids from individual mice were directly measured from immunoblots using a secondary antibody conjugated to the infrared fluorescent dye Alexa Fluor 680 and the Odyssey Infrared Imaging System. The advantage of this system is that the fluorescent intensity of a band is directly proportional to serpina1 concentration (Fig. 2 E, see standard curve). Large amounts of serpina1 (54.3 ± 1.3 picomole [2.9 µg]/femur) were measured in steady-state BM fluids (Fig. 2 C), dropping to 11.0 ± 1.2 picomole [0.5 µg]/femur on day 6 of G-CSF mobilization and rebounding (to 185 ± 49 picomole [9.8 µg]/femur) during the recovery from mobilization. A similar drop in serpina1 concentration (down to 5.17.6 picomole/femur) was observed in the BM of CY-mobilized mice at days 610 during mobilization, with serpina1 levels again rebounding to 6166 picomole/femur on days 1214 (Fig. 2 D).
As no specific antibodies for murine NE and CG are available, it was not possible to directly measure changes in protease concentration during mobilization. Instead we measured proteolytic activity using specific chromogenic substrates and then calculated the molar concentration of each within a femur using purified human NE and CG as standards (Fig. 2, C and D). As serpina1 inhibits both NE and CG with a 1:1 stoichiometry (19, 20), the concentration of serpina1 (53 kD) in steady-state BM fluids is sufficient to inhibit all NE and CG present in a steady-state femur. However, at the peak of mobilization, serpina1 decreases and active NE and CG in the BM are in large molar excess (up to 45 times over serpina1 on day 6 of G-CSF and 36 times on day 6 after CY injection).
Serpina1 within the BM may originate from two possible sources. It may derive from blood slowly diffusing through the BM endothelium into the BM extracellular fluid, or alternatively, serpina1 may be endogenously produced by BM cells. If continuous diffusion from blood was the predominant source of BM serpina1, it is likely that this process would continue during mobilization with the "fresh" serpina1 being continuously cleaved and complexed by the increased active neutrophil serine proteases. This would result in the accumulation of cleaved and complexed serpina1 in the BM fluid. Alternatively, a decrease in endogenous production of serpina1 by BM cells during mobilization would also result in the observed decrease in intact serpina1 levels within the BM fluid but without an associated increase in cleaved or complexed serpina1 forms.
We first confirmed the identity of the 50- and 76-kD bands detected on previous blots (Fig. 2, A and B), by incubating mouse plasma with increasing concentrations of NE for 30 min at 37°C (Fig. 3 A). Indeed the 53-kD "intact" serpina1 band disappeared when incubated with increasing concentrations of NE, with a concurrent 3.5-fold increase in the 50-kD band (cleaved serpina1) and sixfold increase in the 76-kD band (complexed serpina1). The concentration of 50-kD cleaved and 76-kD complexed serpina1 bands were then quantified in BM extracellular fluids from G-CSF and CY-mobilized mice (Fig. 3 B). This quantification revealed that the concentration of cleaved and complexed serpina1 did not increase in BM extracellular fluids during mobilization suggesting that serpina1 diffusion from the blood through the BM endothelium is limited and below the rate required to replenish the pool of intact serpina1 cleaved by NE and CG released by BM neutrophils. This conclusion is supported by the fact that when the endothelium barrier is disrupted at day 3 after CY injection (Fig. 2, B and D), serpina1 diffusion into the BM extracellular fluid dramatically rises resulting in an 11.5-fold increased serpina1 concentration compared with nonmobilized mice. Taken together, these data indicate that serpina1 diffusion through the BM endothelial barrier is limited and endogenous production within the BM is likely to be the predominant contributor to the large amounts of serpina1 observed within BM.
|
Using primers that amplify transcripts from the five murine serpina1 genes (serpina1ae), we observed a dramatic decrease in serpina1ae mRNA levels in the BM of mice mobilized with G-CSF (8,000-fold decrease) or CY (790-fold decrease; Fig. 4 A). When primers to ß2-microglobulin (ß2m), another ubiquitously expressed gene, were substituted for the pan-serpina1 primers, there was no difference between saline-, G-CSF, or CY-injected mice. These results confirm that serpina1 genes are transcribed in the BM, and that the concentration of transcripts decreases during mobilization. Furthermore, the decrease in concentration of mouse serpina1ae transcripts is specific (no changes are found with other RNAs such as ß2m) when compared with the vimentin.
|
Real-time RT-PCR was performed from BM at different time-points of G-CSFinduced mobilization. The decrease in serpina1 mRNA on days 2, 4, and 6 (Fig. 4 C) corresponds to the timing of decreased serpina1 protein in BM fluids (Fig. 2) as well as the rise in colony-forming cells mobilized into the peripheral blood (Fig. 4 E).
Similar decreases in serpina1ae mRNA levels were observed in the BM at the peak of CY-induced mobilization (days 68). Together these data indicate that a significant two to three log decrease in serpina1ae mRNA concentration occurs in the BM at the peak of mobilization even when two completely different mobilization protocols (either G-CSF, a cytokine, or CY, a cytotoxic agent) are used.
Transcription of the neutrophil proteases NE, CG, as well as VCAM-1 and CXCR4 in the BM remains unchanged during mobilization
In sharp contrast to serpina1, no significant change in NE or CG transcripts was observed during mobilization induced by G-CSF or CY (Fig. 4 D and Fig. 5). However this is not surprising as these two proteases are transcribed in myeloid progenitors (see Fig. 6 E and reference 25) and stored in azurophil granules where they are released upon granulocyte activation (26) or in response to mobilizing agents (27).
|
|
Serpina3 mRNA decreases in the BM at the peak of mobilization
Using the same approach, we followed the mRNA levels of six murine serpina3 genes that encode serpins with an NH2-terminal secretion sequence and thus are potentially secreted by cells (serpina3b, serpina3c, serpina3d, serpina3k, serpina3m, and serpina3n; 23). A similar decrease in transcripts (between 8- and 1,400-fold) was found in the BM at the peak of mobilization induced by either G-CSF or CY (Fig. 5, bottom) with the exception of serpina3b, which was not transcribed in the BM of BALB/c mice. We also analyzed by real-time RT-PCR, transcripts of another five murine serpina3 genes that have no secretion sequences and are thus presumed to remain cytoplasmic (serpina3e, serpina3f, serpina3g, serpina3h, and serpina3i), the majority of which were detected in the BM of steady-state BALB/c mice (Table I). Similar to the secreted serpina3, the mRNA levels of these other serpina3 genes were significantly decreased at the peak of mobilization (-fold reduction in BM transcription at day 4 of G-CSF was 85 with P < 0.001 for serpina3e and serpina3i, 621 with P < 0.01 for serpina3g, and 1,592 with P < 0.01 for serpina3h). Serpina3f was not expressed in steady-state or mobilized BALB/c BM.
|
We first examined myeloid cells as they are the predominant cell type accumulating in the BM at the peak of mobilization (Fig. 6 A). Bone marrow myeloid cells were sorted on the basis of positive CD11b (Mac-1) expression and relative maturity on the basis of Gr-1 expression (Fig. 6, B and C) with primitive-mono/myeloid cells being Gr-1dim (sort gate R3), immature myeloid intermediate for Gr-1 staining (sort gate R4), and mature BM neutrophils Gr-1bright (sort gate R2). With G-CSFinduced mobilization a twofold increase in both the proportion and total cell number of primitive Mac-1+Gr-1dim/intermediate myeloid cells (R3 plus R4 regions) was found in the BM.
By real-time RT-PCR, serpina1ae mRNA transcripts were greatest in BM neutrophils (Mac-1+ Gr-1bright) with one or two log less mRNA in more primitive Gr-1intermediate and Gr-1dim cells, respectively (Fig. 6 D). Importantly, at the peak of G-CSF mobilization, a dramatic and significant transcriptional down-regulation of serpina1ae genes was observed when compared with saline-injected BM in all myeloid subsets. This transcriptional down-regulation was not just specific to serpina1ae genes, but was also found with the secreted 1-antichymotrypsin serpina3c (mRNA concentration was 5-fold lower in BM neutrophils and 10-fold lower in more primitive myeloid cells) during mobilization. However, no change was found for other mRNAs transcribed by these cells during mobilization. For example, although transcription of the protease CG is higher in more primitive myeloid cells than in mature BM neutrophils (consistent with packaging in primary azurophil granules), administration of G-CSF was not found to alter CG transcript levels in any of the three myeloid cell populations sorted from the BM (Fig. 6 E).
A similar 1001,000-fold decrease in serpina1ae mRNA levels was also observed during mobilization for other sorted BM cell populations such as B220+ B lymphocytes, CD4+ and CD8+ T lymphocytes, and total CD45+ hematopoietic cells (unpublished results).
Decreases in serpina1 transcripts is restricted to the BM of mobilized mice
During mobilization, a large number of the mobilized HPCs home to the spleen. We therefore queried whether the drop in serpina1 and serpina3 transcripts during mobilization was a general phenomenon or restricted to the BM. For this purpose, we analyzed serpina1 mRNA levels in liver, BM, and spleen by real-time RT-PCR and confirmed these results at the protein level by immunohistochemistry of tissue sections.
SERPINA1 and SERPINA3 proteins are present in the blood at high concentrations at 1.5 and 0.5 mg/ml, respectively, in humans (28). They are mainly produced by the liver and released into the blood. The regulation of expression has not been exhaustively studied in the mouse. As expected, we found high levels of serpina1ae mRNAs (Fig. 7 A, left) and serpina1 protein expression (Fig. 7 B, left) in liver cells, with no change in mRNA or protein expression during G-CSFinduced mobilization. Conversely, serpina1ae transcripts dropped dramatically (1,000-fold) in mouse BM during G-CSFinduced mobilization (Fig. 7 A) with a corresponding sharp drop in protein levels (Fig. 7 B, middle).
|
RT-PCRs using primers for serpina3c revealed a similar phenomenon, that is a drop in mRNA levels in the BM and increased mRNA concentrations in spleen during mobilization (Fig. 7 A, right). In the liver, mobilization also resulted in a 10-fold decrease in serpina3c transcripts.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interestingly, hepatic transcription of serpina1 was not altered during HPC mobilization, resulting in constant blood plasma levels. This would explain in part why there is no active NE or CG in the blood and no cleavage of VCAM-1 on the lumen of BM sinuses that are in direct contact with blood (9). VCAM-1 expression in the spleen does not change during mobilization (9). In this study using immunohistochemistry as well as real-time RT-PCR, we show that cells producing serpina1 proteins accumulate in the spleen during mobilization, possibly protecting VCAM-1 from proteolytic cleavage by neutrophil serine proteases in this tissue.
The human genome has only one copy of the SERPINA1 and SERPINA3 genes and their target protease specificity is well established (29). Both human SERPINA1 and SERPINA3 proteins can inhibit NE and CG in vitro, however human SERPINA1 preferentially targets NE (with the reactive center loop P1P1' amino acids "MS"; 19) whereas SERPINA3 preferentially targets CG (with reactive center loop P1P1' amino acids "LS"; 20). The reactive center loop of many of the murine serpina1 and serpina3 proteins have diverged (30), although several still exhibit the canonical 1-antitrypsin reactive center loop (murine serpina1a, serpina1b, serpina3b, and serpina3n) whereas others have the canonical
1-antichymotrypsinreactive center loop (murine serpina1e, serpina3c, serpina3d, and serpina3j; Table I). Therefore members of both the murine serpina1 and serpina3 families are likely to be involved in the inhibition of serine protease activity in steady-state BM as shown in Fig. 1.
An interesting finding of this study is that although the level of serpina1ae transcripts dropped dramatically in the BM of mice during mobilization, hepatic mRNA levels did not alter resulting in no changes in plasma serpina1 concentrations. Earlier studies on human SERPINA1 transcription/expression reveal five alternative transcription starts can be used depending on the tissue in which expression occurs (with three transcription start sites in leukocytes, one in cornea and one in hepatocytes; 31). In this study we found the reduction in serpina1 transcripts was specific to BM leukocytes during mobilization whereas hepatic transcription remained unchanged. Although we cannot exclude mechanisms involving mRNA destabilization, this suggests that different tissue-specific serpina1 transcriptional start sites are used by murine leukocyte and/or hepatic cells. In contrast to SERPINA1, the human SERPINA3 gene appears to use a single promoter regardless of tissue-type in which it is expressed (31).
Both human SERPINA1 and SERPINA3 genes are reported to be "acute phase proteins" as expression increases two- to fivefold in the liver during inflammation leading to increased levels of SERPINA1 and SERPINA3 proteins in plasma (32, 33). Whether a similar increase in the transcription of hepatic murine serpina1 occurs during inflammation remains controversial (34, 35). A few studies have reported that several cytokines (such as IL-6, IL-1ß, or TNF-) increase SERPINA1 transcription in human monocytes (36, 37). However, to our knowledge, no down-regulation of human or mouse SERPINA1 or SERPINA3 in response to cytokines/growth factors has been reported previously in leukocytes or even hepatocytes.
An intriguing finding of this study is that the same murine BM Gr-1+ myeloid cells contain large amounts of serine proteases as well as large amounts of serine protease inhibitors, which would suggest that the serpins and proteases, although present in the same cells, are stored in different compartments. This is likely to be the case as serpina1 transcription is greatest in BM neutrophils, whereas serine protease (NE and CG) transcription was highest in primitive mono/myeloid cells (Fig. 6). It is known that in granulocyte precursors, the timing of transcription determines in which granules the protein products are stored (25, 38). For instance, the serine proteases synthesized by primitive myeloid cells are packaged predominantly in primary azurophil granules, whereas granule proteins synthesized by more mature BM neutrophils are more likely packaged in secondary or tertiary granules (39). The release of these distinct granules by a neutrophil occurs at different stages during the inflammatory process (26), so although contained within the one cell, both the serine proteases and their specific inhibitors may be released separately. Alternatively, serpins may be constitutively secreted with the proteases remaining sequestered in granules.
In conclusion, our study is the first to suggest a novel role of serpina1 and serpina3 proteins in regulating the homeostasis of the hematopoietic microenvironment, which ultimately determines the fate of hematopoietic stem cells. This conclusion is strengthened by other studies using genetic linkage analysis showing that the Aat locus containing the serpina1 genes, is significantly linked to BM cellularity in inbred mouse strains (40). As reported previously, many proteoglycans (41), extracellular matrix proteins (4244), cytokines, and chemokines (45, 46) expressed in the BM hematopoietic microenvironment and playing a major role in determining the fate of hematopoietic stem cells, are substrates of the neutrophil serine proteases that these serpins inhibit. Similarly, an earlier report has shown that the addition of either recombinant human SERPINA1 or antileukoproteinase to serum-free culture media boosts in vitro proliferation and survival of HPCs, probably by protecting cytokines from the proteases released by differentiated leukocytes (47). Therefore we propose that (a) serpina1 and serpina3 proteins play a major role in maintaining the molecular and functional integrity of the BM hematopoietic microenvironment, (b) the stoichiometric balance between these serpins and neutrophil serine proteases is inverted locally in the BM during systemic administration of G-CSF or chemotherapy, and (c) the reversal of the serpinprotease balance allows proteases to evade their naturally occurring inhibitors, accumulate in active form and to contribute to the mobilization of HPCs.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peripheral blood was collected in EDTA. Plasma was separated and red cells lysed with 10 mM NaHCO3, pH 7.4, and 150 mM NH4Cl. Nucleated cells were plated in double-layer nutrient agar clonogenic cultures (1,500 cells/dish) in the presence human IL-1, mouse IL-3, mouse CSF-1, and rat KIT ligand as described previously (9). Colonies were scored after a 2-wk culture at 37°C in the presence of 5% O2, 10% CO2, and 85% N2.
The BM content of one femur from each mouse was flushed into 1 ml PBS on ice. After centrifugation for 5 min at 400 g, the supernatant fraction (BM extracellular fluid) was removed and used directly in Western blots or for the digestion of recombinant human VCAM-1. The BM cell pellet, as well as the liver and spleen were used for RNA extraction, immunostaining and cell sorting, or to generate frozen sections.
Flow cytometry and cell sorting.
Bone marrow and spleen cells were stained using 1 µg/ml of directly conjugated mAb (BD Biosciences) for myeloid cells (Gr-1-FITC and CD11b-PE), for B lymphocytes (B220-PE), for T lymphocytes (mix of CD3-FITC, CD4-FITC, CD5-FITC, and CD8a-FITC), and for total hematopoietic cells (CD45-PE). Cells positive for each category were sorted and RNA extracted as described below.
RNA extraction and quantitative real-time RT-PCR.
Total RNA was extracted using Trizol (Invitrogen). After DNase treatment and reverse transcription using random hexamers, quantitative real-time PCR with SYBR green (ABI Systems) was performed after manufacturer's instructions using the oligonucleotide combinations shown in Table I. RNA levels were standardized by parallel RT-PCRs using primers to two different house-keeping genes, vimentin (a cytoskeletal protein, forward primer 5'-CACCCTGCAGTCATTCAGACA-3'; and reverse 5'-GATTCCACTTTCCGTTCAAGGT-3') and ß2-microglobulin (forward primer 5'-TTCACCCCCACTGAGACTGAT and reverse 5'-GTCTTGGGCTCGGCCATA-3'). Additional RT-PCRs for the serine proteases NE (forward primer 5'-ACCCTCATTGCCAGGAACTTC-3' and reverse 5'-CCTGCACTGACCGGAAATTT-3'), CG (forward primer 5'-AGGCAGGGAAGATCATTGGA-3' and reverse 5'-TGGATCAGAAGAAATGCCATGT-3') as well as VCAM-1 (forward primer 5'-CTGGGAAGCTGGAACGAAGTA-3' and reverse 5'-GCCACTGAATTGAATCTCTGGAT-3') and CXCR4 (forward primer 5'-GGCTGACTGGTACTTTGGGAAA-3' and reverse 5'-CCGGTCCAGGCTGATGAA-3') were also performed. A PCR from each sample before reverse transcription was also performed to confirm the absence of contaminating genomic DNA.
VCAM-1 cleavage and immunoblotting.
BM extracellular fluids collected from three mice at day 4 of G-CSFinduced mobilization were pooled and mixed with an equal volume (10 µl) of either pooled BM fluids from three nonmobilized mice or with PBS. Where indicated, protease inhibitors were added at the following concentrations: 1 mg/ml human 1-antitrypsin (Sigma-Aldrich), 1 µM BB-94/Batimastat (British Biotech Pharmaceuticals), 1 mM O-phenantroline. After a 20-min incubation at room temperature, 10 ng recombinant human VCAM-1 (98-kD glycosylated extracellular domain; R&D Systems) was added and the mixture reincubated at 37°C for 20 min. An equal volume of loading buffer (125 mM Tris-HCl, pH6.8, 20% glycerol, 2% SDS) was then added and samples boiled for 3 min, before separation by electrophoresis on 10% SDS-PAGE and transfer onto nitrocellulose membrane. Membranes were immunoblotted using a goat antihuman VCAM-1 antibody as described previously (9, 10).
Immunoblotting for serpina1 and 2-macroglobulin, and measurements of serpina1 concentration.
10 µl aliquots of pooled BM extracellular fluids at indicated time-points of G-CSF or saline administration were mixed with an equal volume of loading buffer with 10 mM dithiothreitol, boiled for 3 min, and separated by electrophoresis on 10% SDS-PAGE. After transfer onto nitrocellulose membranes and blocking in Odyssey blocking buffer, membranes were incubated for 1 h with an antihuman 1-antitrypsin (SERPINA1) Ab cross-reacting with mouse serpina1 diluted 1/3,000 in Odyssey blocking buffer with 0.5% Tween 20. After extensive washes with PBST, membranes were incubated with a 1/3,000 dilution of Alexa Fluor 680conjugated goat antirabbit IgG (Molecular Probes). Visualization and quantification were performed on the Odyssey Infrared Imaging System (LI-COR Biosciences) equipped with two solid phase lasers at 680 and 800 nm with a resolution of 169 µm. The integrated intensity of the serpina1 band in the samples was compared with that of mouse plasma serpina1 and the moles per femur calculated, assuming the concentration of murine serpina1 in blood to be the same as in humans, 1.5 mg/ml (28), with a molecular weight of 53,000. Some experiments were performed with BM extracellular fluids from three individual mice from each time-point group and average concentration ± SD were calculated.
Immunoblotting for 2-macroglobulin were performed by incubating the membranes with a 1/3,000 dilution of a rabbit antihuman
2-macroglobulin (Sigma-Aldrich) followed by an incubation with a 1/10,000 dilution of a horseradish peroxidaseconjugated donkey F(ab)'2 antirabbit IgG (Jackson ImmunoResearch Laboratories). Blots were revealed by enhanced chemoluminescence.
Measurement of protease catalytic activity.
The concentrations of active NE and CG in BM extracellular fluids were measured using the chromogenic substrates methyl-O-succinyl (MetOSuc)Ala-Ala-Pro-Valparanitroanilide (pNA) and Suc-Ala-Ala-Pro-Phe-pNA (Calbiochem-Novabiochem) as described previously (9, 10). Measurements were calibrated using serial dilutions of purified human NE and CG (Elastin Products) and molar concentrations were calculated using molecular weight of 30,000 and 29,000 for murine NE and CG, respectively.
Immunohistochemistry.
Frozen sections of mouse liver and spleen were air dried and then fixed for 30 min in methanol containing 0.3% H2O2 to destroy endogenous peroxidases. Slides were then blocked in 4x SSC containing 5% BSA, 5% skim milk powder, and 0.05% Triton X-100 for 2 h before incubation with either 25 µg/ml purified rabbit antihuman 1-antitrypsin or nonimmune-purified rabbit IgG in PBST with 5% skim milk and 5% BSA overnight at 4°C. After extensive washes, slides were incubated with a 1/500 dilution of horseradish peroxidaseconjugated donkey F(ab)'2 antirabbit IgG with minimal cross-reactivity to mouse proteins for 2 h at room temperature, and then washed. Staining was revealed using 0.5 mg/ml 3,3' diaminobenzidine in 50 mM Tris-HCl, pH 7.4, 0.3% H2O2 before counterstaining with hematoxylin and mounting with Aquamount (BDH).
For BM wax-embedded sections, femoral BM was fixed by perfusing 0.05% glutaraldehyde, 2% paraformaldehyde into the descending femoral aorta and femurs processed exactly as described by Nilsson et al. (48). Longitudinal 3.5-µm sections were cut, dewaxed in xylene, and then rehydrated using progressive baths from ethanol to water. After antigen retrieval (12 min boiling in 10 mM sodium citrate, pH 6.0), endogenous peroxidase elimination and antigen labeling were performed as described above.
Statistics.
Levels of significance were calculated using the nonparametric Mann-Whitney test.
![]() |
Acknowledgments |
---|
This work was supported by grants 080193 and 288701 (J.-P. Lévesque) from the National Health and Medical Research Council of Australia. Paul Coughlin is a Wellcome Trust Research Fellow.
The authors have no conflicting financial interests.
Submitted: 8 November 2004
Accepted: 25 January 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|