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Blood, 15 September 2006, Vol. 108, No. 6, pp. 1857-1864. Prepublished online as a Blood First Edition Paper on May 30, 2006; DOI 10.1182/blood-2005-10-007658.
HEMATOPOIESIS
Adult murine hematopoiesis can proceed without
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4
1 and 47 integrins in hematopoiesis is controversial. While some experimental evidence suggests a crucial role for these integrins in retention and expansion of progenitor cells and lymphopoiesis, others report a less important role in hematopoiesis. Using mice with a deletion of the 1 and the 7 integrin genes restricted to the hematopoietic system we show here that 41 and 47 integrins are not essential for differentiation of lymphocytes or myelocytes. However, 17 mutant mice displayed a transient increase of colony-forming unit (CFU-C) progenitors in the bone marrow and, after phenylhydrazine-induced anemia, a decreased number of splenic erythroid colony-forming units in culture (CFUe's). Array gene expression analysis of CD4+CD8+ double-positive (DP) and CD4–CD8– double-negative (DN) thymocytes and CD19+ and CD4+ splenocytes did not provide any evidence for a compensatory mechanism explaining the mild phenotype. These data show that 41 and 47 are not required for blood cell differentiation, although in their absence alterations in numbers and distribution of progenitor cells were observed. |
Introduction |
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and a subunit.1 Integrins provide mechanical support by connecting the extracellular matrix (ECM) with the cytoskeleton, but are also capable of transducing chemical signals upon ligand binding. This signaling results in cytoskeleton reorganization and changes in gene expression affecting proliferation, differentiation, and survival of cells.2 Molecules inside the cell, on the other hand, can modulate the affinity and avidity of integrins, called inside-out signaling, which is, for example, crucial for the extravasation of leukocytes.3
In vitro and in vivo experiments suggests an important role of 41 and 51 integrins for the adhesion of HSCs and hematopoietic progenitor cells (HPCs) to fibronectin in the bone marrow matrix.4,5 Integrin 41 was additionally shown to mediate binding to VCAM-1, which is expressed on BM stroma cells.6 Injection of fibronectin fragments and blocking antibodies against 41 and VCAM-1 led to a release of HSCs/HPCs into the blood, supporting the proposed importance of these interactions in vivo.5,7 Conditional deletion of the VCAM-1 gene resulted in an early exit of B-cell precursors into the blood.8 Finally, it was shown that 41–mediated attachment of HPCs to fibronectin promotes proliferation and survival,9,10 suggesting a crucial role for self-renewal and survival of HSCs.
In vivo studies with 1 integrin–deficient somatic chimeric mice, which are generated by injecting 1-null embryonic stem (ES) cells into wild-type host blastocysts, demonstrated that 1 integrin is not required for the formation of HSCs, but is essential for their migration to the fetal liver.11 Additionally, 1 integrin–deficient HSCs failed to engraft lethally irradiated mice.12 Altogether, these data pointed to a key role of 41 integrin in hematopoiesis. This notion was corroborated by the analysis of 4-null somatic chimeric mice, which have almost no mature B cells, T cells, or erythroblasts derived from 4-null ES cells.13,14 In vitro experiments with cells derived from the 4-null chimeric mice suggested that both erythroid and B-cell precursors are less able to transmigrate through the stroma, which may result in reduced cell proliferation.14 Also, the number of 4-deficient myeloid cells was reduced compared with control chimera. Since 7 integrin constitutive null mice displayed normal hematopoiesis,15 it was suggested that 41 integrin might be the pivotal integrin during hematopoiesis, as 4 can dimerize only with 1 and 7 integrins. Therefore, it was unexpected when 1 mutant BM chimeras showed no defects in blood cell development.16 The simplest explanation at that time was that 41 and 47 integrin might have redundant functions in blood cell development and that only the absence of both receptors leads to the described hematopoietic defects. However, further experiments showed that inducible deletion of the 4 integrin gene has only subtle effects on hematopoiesis.17 These mutants showed only a partial reduction of the B220+ B-cell and CD4+ T-cell populations in BM. Monocytes (Mac-1+) and erythroblasts (Ter119+) were reported to occur in normal amounts in the BM. In this study, however, the 4 integrin gene was not only deleted in hematopoietic cells but also in many nonhematopoietic cells such as hepatocytes, endothelial cells, and so on, which could contribute to the phenotype. An alternative explanation for these contrasting results could be that fetal hematopoiesis is more dependent on 4 integrin than adult hematopoiesis.
To better understand the role of 41 and 47 integrin in adult hematopoiesis, we generated and analyzed mice with a blood cell–restricted knockout of 1 and a constitutive knockout of 7 integrin. As a consequence 47, 41 and also other 1 integrins expressed on blood cells are lost. In contrast to the 4-null somatic chimeras13,14 or the 4 conditional knockout mice17 used previously, we can exclude any effects due to deletion of 4 on nonhematopoietic cells, which might influence hematopoiesis through altered production of cytokines and growths factors or different cell-cell interactions. This model was used to study HSC maintenance, HPC distribution and differentiation, and the migration of differentiated cells in the absence of 1 and 7 integrins in adult mice. We demonstrate now that even in the absence of both 41 and 47 integrins, hematopoiesis is normal.
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Materials and methods |
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1 and the 7 integrin genes in the hematopoietic system
Mice carrying a 1 integrin gene flanked by loxP sites (fl/fl)16 were mated with mice with a neomycin-disrupted 1 integrin gene (+/–),18 mice lacking a functional 7 integrin gene (7–/–),13 and with mice carrying the Mx transgene (+Mx).19 17 mutant BM chimeras were generated by BM transplantation as described previously.20 Recipient Ly-5.1+ mice were lethally irradiated and received BM cells from Ly-5.2+ 1fl/+7–/–+Mx or 1fl/fl7–/– mice (7 mutant BM chimeras) or from 1fl/fl7–/–+Mx mice (17 mutant BM chimeras). Four weeks after the transfer, deletion of the 1 gene was induced by polyIC injections as described previously.20
Animal treatment
Mice were maintained and bred under pathogen-free conditions. All animal experiments were approved by the local ethics committee. Blood samples were obtained from the retro-orbital plexus under anesthesia. Acute hemolysis was assessed after phenylhydrazine (PHZ; Sigma, Steinheim, Germany) treatment as described.16
Türk staining
Whole blood of control and 17 mutant BM chimeras was isolated, diluted 1:10 with Türk stain (0.01% gentian violet, 1.0% acetic acid), and differentially counted for polymorphonuclear and mononuclear cells in a hemocytometer.
Flow cytometry
Single-cell suspensions were prepared and analyzed as described.16 Erythrocytes in blood samples were lysed by incubation in ACK-lysis buffer for 5 minutes at room temperature prior to staining.21
Deletion of the 1 integrin gene on BM stroma cells was assessed by measuring the activity of the -galactosidase reporter.16 Five days after a single injection of 250 µg polyIC, BM cells were plated on tissue-culture plates as described.22 After 24 hours, nonadherent cells were removed and adherent cells detached by trypsin/EDTA. Nonhematopoietic BM stroma cells were characterized as Ly-5.2–Ter119–adherent cells, which consist of mesenchymal stem cells, fibroblasts, endothelial progenitor cells, and endothelial cells.23 Hematopoietic cells, on the other hand, were identified as Ly-5.2+ or Ter119+ nonadherent cells. Cells were stained for -galactosidase activity as described,24 with minor changes. Briefly, 4 x 106 cells were suspended in 20 µL phosphate-buffered saline (PBS) added to 20 µL of 2 mM fluorescein-di-(beta-D-galactopyranoside) (FDG; Sigma). Cells were incubated at 37°C for 75 seconds and subsequently 200 µL ice-cold PBS was added. Cells were incubated for 3 hours on ice and analyzed by flow cytometry as described.16
For the analysis of platelets, 5 µL antibody solution containing FITC-conjugated anti–1 integrin (Ha2/5; 1:10 diluted; BD Pharmingen, San Diego, CA) and PE-conjugated anti–GPIb-IX (p0p1) (kindly donated by Dr B. Nieswandt, University of Würzburg, Germany; 1:10 diluted) was added to 1 µL whole blood. After a 15-minute incubation at room temperature in the dark, 100 µL PBS was added and samples were analyzed by fluorescence-activated cell sorting (FACS).
Colony formation assay
Pre-B and CFU-C colony formation assays were performed as described previously.16 CFUe assays were carried out following the instructions of the manufacturer (Stem Cell Technologies, Vancouver, BC, Canada).
Separation of splenocytes by MACS
Leukocyte subpopulations were isolated from single-cell suspensions of splenocytes by positive selection using FITC-conjugated antibodies against B220 (B cells), CD4 (CD4 T cells), or CD8 (CD8 T cells) and anti-FITC MACS beads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the sort was checked by FACS analysis.
Southern blot analysis
Southern blot analysis was carried out as described.16 Membranes were exposed to x-ray films and the resulting bands quantified using Bio-PROFIL Bio-1D V97.03 software (Vilber Lourmat, Marne-la-Vallée, France).
DNA microarray hybridization and analysis
Total RNA was isolated from FACS-sorted populations of thymocytes (DN, CD4–CD8–; DP, CD4+, CD8+) and splenocytes (CD19+ B cells; CD4+ T cells). For biotin-labeled target synthesis reactions were performed using standard protocols supplied by the manufacturer (Affymetrix, Santa Clara, CA). Briefly, 5 µg total RNA was converted to double stranded (ds) DNA using 100 pmol of a T7T23V primer (Eurogentec, Seraing, Belgium) containing a T7 promotor. The cDNA was then used directly in an in vitro transcription reaction in the presence of biotinylated nucleotides.
The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 µg of each biotinylated cRNA preparation was fragmented and placed in a hybridization cocktail containing 4 biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MOE430A for 16 hours. After hybridization, the GeneChips were washed, stained with SA-PE, and read using an Affymetrix GeneChip fluidic station and scanner. Gene expression levels were determined by means of Affymetrix's Microarray Suite 5.0 (MAS 5.0).
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Results |
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1 and 7 integrins are coexpressed in many hematopoietic cells, including HSCs
In order to replace each other functionally, 41 and 47 must be expressed in the same cells. While 1 integrin is expressed on all hematopoietic cells besides erythrocytes,12,16 the expression of 7 integrin is more restricted (Figure S1, available on the Blood website; see the Supplemental Figure link at the top of the online article). In BM, 7 integrin was found on lin–c-kit+Sca1 high cells (ie, bona fide stem cells), most mature B cells (B220 high), on subpopulations of mature and immature granulocytes (Gr-1 high; Gr-1 medium), and on few erythroid cells (Ter119+) and immature B cells (B220 low). 7 integrin was furthermore found on subsets of DN, CD4SP, and CD8SP thymocytes, whereas it was virtually absent on DP thymocytes. In spleen and lymph nodes, 7 integrin was present on most B cells (B220+), T cells (CD4+, CD8+), and granulocytes (Gr-1+). In lymph nodes, about 50% of the erythroid cells (Ter119+) expressed 7 integrin, whereas only a few percent of the erythroid cells in the spleen had 7 on their surface.
Normal maintenance of HSCs in the combined absence of 1 and 7 integrins
To directly assess possible redundant functions of 41 and 47, mice were generated lacking both receptors in the hematopoietic system. Mice carrying a conditional knockout for 1 integrin, a 1-null allele, and a cre recombinase transgene under the control of the polyIC-inducible Mx-promotor were intercrossed with mice lacking a functional 7 integrin gene.15 Thus, mice that were deficient for 7 integrin and carried an inducible 1-null gene (1fl/–7–/–Mx-cre+ or 1fl/fl7–/–Mx-cre+) were obtained. Mice lacking 7, but constitutively expressing 1 (1fl/+7–/–Mx-cre+ or 1fl/fl7–/–) were used as controls. To restrict the deletion to the hematopoietic system, BM from these mice was transplanted into lethally irradiated recipient mice (Figure 1A). The ablation of the conditional 1 gene was induced by 3 intraperitoneal injections of polyIC after reconstitution of the hematopoietic system (4 weeks after irradiation). Mice were analyzed 2, 6, and 10 to 12 months after the polyIC treatment.
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7 integrin was detectable in any tissue by FACS (data not shown). To analyze the time course of the 1 integrin gene ablation, we monitored the loss of 1 integrin expression on short-lived platelets. Two days after the first polyIC injection, 1-deficient platelets were already detectable in the blood of 17 mutant BM chimeras (Figure 1B). The relative amount of 1-deficient platelets increased continuously to reach 93% after 14 days and 97% after 21 days and later. In control BM chimeras, on the other hand, virtually all platelets expressed 1 integrin at all time points analyzed. These data show that the deletion of the 1 integrin gene can be induced within a few days in a 7 mutant background. Furthermore, they confirm that the development of megakaryocytes and platelets is not crucially dependent on 1 and 7 integrins. Southern blot analysis of BM, spleen, and thymus of 2- and 10-month-old 17 mutant and control BM chimeras confirmed the efficient 1 gene deletion in all these tissues (Figure 1C and data not shown). Since only HSCs can sustain hematopoiesis for more than 3 months, these data indicate that 17-deficient HSCs are maintained in vivo.
To investigate the development of different hematopoietic lineages that derive from HSCs, we first checked the cellularity of different lymphoid organs. At 2 months (Figure 1D, left) and 10 to 12 months (data not shown) after induction of the gene deletion there were no differences observed in the cellularity of BM, thymus, or spleen of control and 17 mutant BM chimeras, providing no evidence for defective hematopoiesis in the absence of 1 and 7 integrins. Differential blood counts revealed similar numbers of mononuclear and polymorphonuclear cells in the peripheral blood (PB) of control and 17 mutant BM chimeras 6 months after polyIC treatment (Figure 1D, right).
Normal B-cell development in the absence of 1 and 7 integrins
Since previous studies suggested that normal B-cell development was dependent on 4 integrin,13,14,17 but not on 4116 or 4715 alone, we investigated whether 1 and 7 integrins have a redundant function in B-cell development. In pre-B colony assays, control and 17 mutant BM gave rise to colonies that were derived each from a single pre-B-cell pecursor. FACS analysis of randomly picked colonies confirmed that 36 of 39 colonies (92.3%) of 17 mutant BM did not express 1 integrins, whereas all tested colonies derived from control BM expressed 1 integrins. No host-derived colonies expressing Ly-5.1 were detected. To further monitor B-cell development, single-cell suspensions from BM, spleen, and lymph nodes (LNs) were analyzed using B-cell–specific markers: B220 (pre-proB and later), CD19 (proB and later), IgM (immature B), and IgD (all mature B). The relative amount of cells positive for the respective markers was unaltered in 17 mutant BM chimeras compared with control BM chimeras 2 and 12 months after the knockout induction (Table 1 and data not shown).
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1 integrin (Figure 2A). Mature B cells (B220 high) express only low amounts of 1 integrin, which makes it difficult to distinguish normal from 1-deficient mature B cells by FACS (Figure 2A). Therefore, the knockout efficiency in B220+ B cells purified from spleen was determined by Southern blot analysis (Figure 2B). B220+ B cells were enriched by MACS beads to a purity of more than 95% (Figure 2B, left). Southern blot of genomic DNA isolated from these cells revealed a deletion efficiency of the 1 integrin gene of 93.5% ± 8.3% (n = 5). These data indicate that in the absence of 1 and 7 integrins, B cells can fully mature. Furthermore, since spleen, LN, and BM contained normal numbers of B cells, migration of immature B cells to spleen and of mature B cells to LN and BM is apparently not impaired by the combined loss of 1 and 7 integrins.
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1 and 7 integrins
Since in 4-null somatic chimeric mice 4-null T-cell precursors were described to be unable to migrate to the thymus for further differentiation, thymocyte development was analyzed in 17 mutant BM chimeric mice using the T-cell markers CD4 and CD8. No significant difference was found in the population sizes of CD4–CD8– (DN) thymocytes, which contain the early thymic immigrants indicating that thymic colonisation was not altered in 17 mutant BM chimeric mice which lack both 41 and 47 integrins. Furthermore, the relative amounts of CD4+CD8+ (DP), CD4+ (CD4SP), and CD8+ (CD8SP) cells in the thymus were normal in 17 mutant BM chimeric mice (Figure 3A and Table 2). Staining of thymocytes for 1 integrin and subsequent FACS analysis proved the absence of 1 integrin from DP T cells (Figure 3B). Normal numbers of CD4 and CD8 T cells in spleen, LN, and BM of 17 mutant BM chimeras 2 and 12 months after induction of the 1 gene deletion suggested normal migration of these cells to secondary lymphoid organs and to the BM (data not shown). Since mature CD4+ and CD8+ T cells express only low levels of 1 integrin, the deletion efficiency in these populations was tested on the genomic level. Southern blot from CD4+ and CD8+ T cells, enriched from the spleen, revealed that 78.5% ± 5% (n = 3) of CD4+ T cells and 83.4% ± 10.8% (n = 4) of CD8+ T cells lacked a functional 1 integrin gene (Figure 3C). These data show that 1 and 7 integrins are neither essential for the migration of T-cell precursors to the thymus nor for T-cell maturation within the thymus.
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1 and 7 integrins
To analyze myeloid development, we first studied the capacity of myeloid progenitors in control and 17 mutant BM chimeras lacking 41 and 47 integrins to form colonies in vitro (CFU-C). All CFU-C colonies analyzed from BM (n = 37), spleen (n = 38), and PB (n = 36) from control mice were positive for 1 integrin. From 17 mutant BM chimeras only 2 of 36 colonies from the BM, 1 of 37 of the spleen, and 3 of 37 colonies derived from PB were positive for 1 integrin. These results show first, that in the absence of 1 and 7 integrin granulocyte/monocyte precursors have the potential to form colonies in vitro and second, that the efficiency of the 1 integrin gene deletion is very high in the myeloid lineage. Both control and mutant BM cells also formed erythroid colonies (CFUe's) in vitro. Of 42 colonies tested from mutant BM, none showed a functional 1 integrin gene as tested by genomic PCR, whereas in 24 of 24 colonies from control BM a functional 1 gene was detected.
Monitoring the development of monocytes, granulocytes, and erythroblasts in 17 mutant BM chimeric mice 2 and 12 months after the 1 integrin gene deletion in vivo revealed no significant differences in the numbers of granulocytes, monocytes, and erythroblasts, indicating no developmental defects in the absence of both 41 and 47 integrins (Table 3 and data not shown). The 1 gene deletion on these cells was confirmed by staining for 1 integrin and subsequent FACS analysis (Figure 4). These data strongly suggest that HSCs and HPCs continuously provide myeloid and erythroid cells in the absence of 41 and 47 integrins.
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Induced deletion of 4 integrin in hematopoietic and many nonhematopoietic cells resulted in a slow increase of CFU-Cs in the BM, an overproportional release into the PB, and an accumulation of CFU-Cs in spleen, suggesting a role for 4 in the retention of progenitor cells in the BM.17 To test this in 17 mutant BM chimeras, we determined the frequency of CFU-C progenitors in BM, PB, and spleen 2 and 10 months after the 1 integrin gene deletion (Figure 5A). At 2 months, the number of precursor cells was significantly elevated in the BM of 17 mutant mice as compared with controls. We also observed an increase of progenitors in PB roughly proportional to the progenitor increase in the BM, but significantly less than reported for 4 conditional knockout mice 8 weeks after induced gene deletion, thus not indicating a severe defect in progenitor retention in the BM. Furthermore, these alterations were transient, since they were observed 2 months but not 10 months after knockout induction, when 17 mutant and control mice had similar CFU-Cs, both in BM and PB (Figure 5A). Unlike the conditional 4 integrin knockout mice 2 weeks and 6 months after gene deletion,17 17 mutant BM chimeras did not accumulate precursor cells over time in the spleen, as tested 2 and 10 months after the knockout induction (Figure 5A). To the contrary, CFU-Cs were significantly decreased in 10-month-old mutant chimera.
FACS analysis of BM cells of nonchimeric (1fl/flMx-cre+) mice 3 days after a single polyIC injection revealed that the 1 integrin gene is not only deleted on most hematopoietic cells (Figure 5B, Ly-5.2+, Ter119+), but also on many nonhematopoietic BM stroma cells, defined as (Ly-5.2–, Ter119–) plastic adherent cells (Figure 5B). To assess whether loss of 1 and 7 integrin on nonhematopoietic cells might contribute to the progenitor release, the frequency of progenitor cells was determined in the PB of nonchimeric 17 mutant mice 4 weeks after the knockout induction. We found that the progenitor content in PB increased approximately 8-fold in 17 mutant mice (data not shown), comparable to the more than 10-fold increase of the CFU-Cs in 4 conditional knockout mice 4 weeks after gene deletion,17 indicating that loss of 4 integrin on nonhematopoietic cells might contribute to the release of CFU-C progenitors from BM to PB.
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After phenylhydrazine (PHZ) induced lysis of erythrocytes in vivo, erythroid precursor cells expand in order to compensate for the loss of erythrocytes. In addition, hemolytic anemia promotes extramedullary erythropoiesis leading to proliferation of progenitors in the spleen.25 Since in 4 conditional knockout mice the ability of erythroblasts to expand in response to a PHZ-induced hemolytic anemia was reduced,17 we investigated whether combined loss of 1 and 7 integrins shows a similar effect. For better comparison with the nonchimeric 4 conditional knockout mice we used nonchimeric 17 mutant mice.
Two days after PHZ treatment the amount of erythrocytes dropped in both control and 17 mutant mice by more than 55% in BM (n = 3) and was not significantly different between both groups. Similarly, also the number of erythroblasts in the BM as assessed by Ter119 staining was reduced after the PHZ treatment but comparable between 17 mutant mice and controls (Figure 6A). Since 4 conditional knockout mice were reported to have fewer erythroid progenitor cells in the BM after hemolytic stress, we tested at the same time point (ie, 2 days after PHZ treatment) the relative amounts of cells of different erythroid developmental stages by Ter119-CD71 staining and subsequent FACS analysis separating different maturation stages of BM erythroblasts18 (Figure 6B). Neither in BM nor in spleen was a significant difference detected between 17 mutant and control mice at any of these stages, providing no evidence for an impaired recovery from hemolytic anemia in the absence of 1 and 7 integrin (Figure 6C and data not shown). Analysis of CFUe's confirmed a normal frequency of erythroid progenitors in BM, but surprisingly revealed a significant reduction of CFUe's in the spleen of 17 mutant mice compared with controls. Since the spleen is the most prominent place for hematopoiesis after PHZ treatment these data support a role for 41 and 47 integrin in the recovery of the erythropoietic system after hemolytic anaemia.
No apparent compensatory change in gene expression in the combined absence of 1 and 7 integrin in different leukocyte subsets
To investigate whether hematopoietic subpopulations of 17 mutant mice show major alterations in gene expression, we tested mRNA levels of different hematopoietic subsets (DN, DP, B cells, CD4+ T cells) by array analysis. RNA was prepared from DP and DN cells from the thymus, and CD19+ (B cells) and CD4+ cells from the spleen, obtained from 5 pooled mutant and control mice, respectively, and tested on affymetrix chips. All mutant mice had an efficient knockout of 1 integrin indicated by a loss of surface 1 integrin on more than 97% of the platelets.
We then analyzed the data by searching for genes that are up- or down-regulated in mutant mice in all 4 different populations investigated, which would suggest a crucial compensatory response. However, only 3 genes encoding heat shock proteins (heat shock protein 1, heat shock protein 1, heat shock protein 105) were found with increased expression in the absence of 1 and 7 integrin. No genes were found with reduced expression in all subpopulations derived from mutant mice.
We then screened the genes up- or down-regulated in the individual hematopoietic subpopulations (thymus: DN, DP; spleen: B cells, CD4+ T cells) for integrins (3-6, 2-10, X, D, M, L, E), selectins (P-, L-, E-), CD44, and for the 41 and 47 integrin ligands VCAM-1 and MAdCAM-1. All these genes showed normal expression in 17 mutant cells compared with control cells.
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Discussion |
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4 integrins, for example, have been suggested to be crucial for the retention of hematopoietic stem cells in the bone marrow, for the homing of lymphocytes to Peyer patches and for the migration of T cells during inflammation.13,15,26 In addition, development of the hematopoietic system, characterized by the formation of the different blood cell lineages and their distribution within hematopoietic organs, was reported to be 4 integrin–dependent, although the gradual contribution of 4 integrins differed significantly depending on the experimental approach.7,17 To study the role of 4 integrins in hematopoiesis, mouse models were applied in which the 4 integrin gene was deleted on hematopoietic as well as on nonhematopoietic cells. Loss of the 4 integrins on the latter cell population might affect hematopoietic development. To overcome this problem and to assess by an alternative approach the function of 4 integrins in hematopoiesis, we decided to generate and analyze mice, which lack 1 and 7 integrins, and hence both 41 and 47 integrins, exclusively in the hematopoietic system. Unexpectedly, we could not find an essential function for 41 and 47 integrins in blood cell development or in progenitor retention in the bone marrow. Detailed analysis of lymphoid and myeloid development by testing the size of different blood cell subsets in lymphoid organs at different time points and investigat
1 and 
