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Previous Article | Next Article 
Molecular and Cellular Biology, February 2003, p. 1034-1043, Vol. 23, No. 3
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.3.1034-1043.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Institute of Experimental Dermatology,1 Institute of Medical Biochemistry,2 Institute of Experimental Pathology, Zentrum für Molekularbiologie der Entzündung,3 Department of Dermatology, Ludwig Boltzmann Institute of Cell Biology, University of Münster, 48149 Münster, Germany4
Received 15 August 2002/ Returned for modification 13 September 2002/ Accepted 22 October 2002
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ABSTRACT |
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INTRODUCTION |
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The intracellular functions of both proteins are less well understood. Several reports have described the association of both proteins with cytoskeletal elements; however, the functional relevance of these observations has so far not been shown (26, 32). Furthermore, it was proposed that both proteins are involved in the terminal differentiation of neutrophils by inhibiting casein kinase (18). The mere abundance of both calcium-binding proteins in neutrophils implies a role in the maintenance of calcium homeostasis in these cells.
Recently, S100A8-deficient mice revealed an unexpected role for this molecule in embryogenesis since the S100A8-deficient embryos were resorbed by day 9.5 (22). The death of these animals at the embryonic stage prevented a detailed functional analysis of S100A8 protein in the adult mouse. Here, we describe experiments with S100A9-deficient mice. These mice appear to be phenotypically normal, even in two inflammatory models. Nonetheless, a detailed analysis of the S100A9-null neutrophils revealed a disturbed G/F-actin balance accompanied by a decreased responsiveness to interleukin-8 (IL-8)-mediated CD11b surface recruitment and a reduced migration upon chemoattractant stimulation in vitro.
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MATERIALS AND METHODS |
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) (IgG2a/k), R35-95; FITC-conjugated rat anti-mouse Ly-6G (Gr1), RB6-8C5; rat anti-mouse CD54R/B220, RA3-6B2; rat anti-mouse Ly-6G (Gr1), RB6-8C5; and rat anti-mouse CD90, Thy-1.2. The following MAbs were also obtained: from BMA (Augst, Switzerland), rat anti-mouse ER-MP20 (T-2004); from Serotec, Ltd., rat anti-mouse F4/80 (A3-1); from Dianova (Hamburg, Germany), peroxidase-conjugated goat anti-rabbit IgG, peroxidase-conjugated goat anti-rat IgG, and FITC-conjugated goat anti-rabbit IgG; and from Miltenyi Biotec (Bergisch-Gladbach, Germany), magnetic-bead-conjugated anti-CD11b. Our laboratory produced the rabbit polyclonal antibodies against mouse S100A8 and S100A9. Recombinant human IL-8 and leukotriene B4 (LTB4) were purchased from TEBU (Offenbach, Germany) and Sigma (Taufkirchen, Germany), respectively. IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) were ordered from R&D Systems (Wiesbaden, Germany). Culture medium. Cell-culture-grade buffers (phosphate-buffered saline [PBS] without Ca2+ or Mg2+, Hanks balanced salt solution [HBSS] with Ca2+ and Mg2+), media (Dulbecco’s modified Dagle’s medium [DMEM], RPMI 1640, and Iscove’s modified Dulbecco’s medium [IMDM]), antibiotics, and sera were purchased from Biochrom (Berlin, Germany).
Targeting construct. The targeting vector (Fig. 1) was constructed by replacing 760 bp of the genomic sequence containing exon 2 and the adjacent intron region with the pMC1-derived neomycin-poly(A) expression cassette (Stratagene, Amsterdam, The Netherlands). This mutation deletes the initial start codon. The resulting construct was linearized with NotI and electroporated into the embryonic stem (ES) cell line E14-1-1 (13) derived from 129/Ola mice. Resistant colonies were isolated following selection in G418 and ganciclovir and expanded as described previously (9).
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To analyze the offspring, PCR from tail DNA was performed using the following primers: upstream, 5'-CTCGGTCAAATGTGCCTAAC, and downstream, 5'-GTCTTTAACCAGGGACTAGG. PCR was performed with a thermal cycler for 10 cycles at 94°C for 90 s, 55°C for 120 s, and 72°C for 390 s, followed by 30 cycles at 94°C for 90 s, 55°C for 80 s, and 72°C for 390 s. Final extension was done at 72°C for 600 s. After PCR amplification of wild-type and targeted alleles, DNA fragments of 4.7 and 5.1 kb were detected.
Isolation of leukocytes. Mouse bone marrow cells were flushed from femurs and tibias with PBS. Erythrocytes were lysed with lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 4 min at room temperature, and the remaining cells were washed once with PBS. Polymorphonuclear neutrophils (PMNs) were isolated by magnetic cell separation using CD11b microbeads (Miltenyi Biotec) according to the manufacturer's instructions. Peripheral leukocytes were purified from heparinized blood from axillary vessels. Neutrophils were analyzed by flow cytometry after specific immunostaining. Thioglycolate-elicited PMNs were harvested as described below.
RNA isolation and detection. Total RNA was isolated from blood, spleen, liver, brain, and thioglycolate-elicited PMNs by using a commercially available reagent (peqGOLD TriFast; peqlab Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer's instructions. Northern blot analysis was performed according to standard procedures. Briefly, total RNA was loaded on a 1% agarose-2.2 M formaldehyde denaturing gel and transferred to a nylon membrane (Hybond; Amersham Bioscience Europe, Freiburg, Germany). Blots were hybridized with 32P-labeled cDNA probes (performed with the HexaLabelDNA labeling kit; MBI Fermentas, St. Leon-Rot, Germany). Total RNA was also reverse transcribed into cDNA with Moloney murine leukemia virus-Superscript II polymerase following the recommendations of the supplier (Gibco BRL via Invitrogen, Karlsruhe, Germany) by using an oligo(dT)12-18 primer (MWG-Biotech, Munich, Germany). Amplification of cDNA was performed with TaKaRa Ex Taq DNA polymerase (BioWhittaker, Verviers, Belgium) and specific oligonucleotides for S100A8 (sense, 5'-GAAATCACCATGCCCTCTAC; antisense, 5'-CCCACTTTTATCACCATCGC), and S100A9 (sense, 5'-CTCTAGGAAGGAAGGACACC; antisense, 5'-GCCATCAGCATCATACACTC). The reaction conditions included 30 cycles of amplification at 94, 55, and 72°C for 90, 120, and 120 s, respectively. Final extension was done at 72°C for 600 s.
Flow cytometric analysis. Immunofluorescence staining was performed after washing the cells once with PBS-0.5% bovine serum albumin (BSA). Cells (106) were first incubated with each anti-MAb (5 µg/ml) diluted in PBS-BSA for 30 min on ice. After three washes with PBS, cells were incubated with FITC- and phycoerythrin-conjugated second-step antibody for 30 min on ice. After washing twice with PBS, the cells were analyzed by flow cytometry with FACStar (Becton Dickinson). For the detection of S100A8 and S100A9, fixation and permeabilization were performed in acetone before intracellular staining.
Immunohistochemistry. Cryostat sections (5 to 8 µm thick) and cytospin preparations were fixed in acetone for 10 min at room temperature and washed in PBS. Endogenous peroxidase activity was blocked by incubating the preparations in sodium azide (Sigma) diluted at 1g/150 ml of PBS plus 0.035% H2O2 for 20 min at room temperature. PBS-1% BSA was used to block nonspecific binding for 60 min. Preparations were stained with primary antibody, followed by incubation with peroxidase-conjugated goat anti-rat or anti-rabbit IgG in PBS-1% BSA-20% inactivated mouse serum for 60 min. Enzymatic color development was performed by using 3-amino-9-ethylcarbazole (Sigma) as substrate, which was diluted in acetate buffer with 0.014% H2O2 at 37°C for 12 min. Slides were then counterstained with Giemsa stain and mounted with Aquamount mounting medium (BD PharMingen).
Colony-forming assay. Bone marrow cells were held in IMDM with antibiotics and 20% fetal calf serum (FCS). One hundred thousand cells were seeded into one well of a six-well dish in 1 ml of medium containing 0.35% low-melting-point agarose (SeaPlaque; Biozyme) and 20 ng of the indicated cytokine. After 10 days of incubation, colonies were counted (bone marrow cell numbers per colony were >50 for cells from S100A9-deficient mice versus >20 for cells from wild-type mice which responded to G-CSF).
PMN emigration into the skin and peritoneum. IL-8 (40 ng in 8 µl of PBS) or PBS was injected subcutaneously into both ears of the mice. Four hours after injection, the skin at the injection site was excised, embedded, frozen in N2, sectioned at an 8-µm thickness, fixed in acetone, and stained in two steps with rat anti-mouse Ly-6G (Gr1) and a peroxidase-conjugated anti-rat IgG antibody (Fig. 12). To evaluate emigration, the Gr1-positive cells were counted in five fields of vision in five sections per ear. Five mice of both the S100A9-deficient and wild-type genotypes were used for the experiment.
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TEM. Transendothelial migration (TEM) was determined by using a chemotaxis microchamber (Transwells, 5-µm pore size; Corning). Bone marrow cells were suspended at 2 x 106 cells/ml in DMEM supplemented with 10% FCS. One hundred microliters of cell suspension was applied to the upper chamber on a monolayer of bEnd5 cells (2 x 104/200 µl of DMEM, 10% FCS; day 3). Cytokines, IL-8 (1 µg/ml), and LTB4 (25 nmol) were added to the lower chamber. After incubation at 37°C for 2 h, cells that migrated to the lower chamber were collected and counted with Coulter (Krefeld, Germany) Counter Z2. Cytospin preparations of the migrated cells were then immunocytochemically stained to determine the proportions of the cell subpopulations.
Measurement of cell migration within a 3-D collagen matrix. Cell migration within a three-dimensional (3-D) collagen matrix was studied as described previously (7, 8). Five hundred thousand neutrophils (isolated from the peritoneal cavity 4 h after the induction of peritonitis) were suspended in 20 µl of medium (RPMI plus 10% FCS) and added to 40 µl of a solution containing dermal bovine collagen type I (Nutacon, Leimuiden, The Netherlands). The final concentration for collagen was 1.6 mg/ml. This suspension was used to fill a self-constructed chamber consisting of a basal microscope slide and an upper coverslip sealed on three sides with paraffin and petroleum jelly at a ratio of 1:1. After polymerization (37°C, 5% CO2, 20 to 30 min), the chamber was filled with medium and basal cell migration was recorded by time lapse video microscopy for 2 h. Replacing the medium with fresh medium containing 500 ng of IL-8/ml stimulated thereafter the locomotion of cells. Cell migration was then further documented over a period of 1 day. Subsequently, the paths of 40 individual cells were reconstructed from the recorded films for a period of up to 4 h by computer-assisted cell tracking (8). For data analysis, 40 cells of each sample were randomly selected. Subsequently, the movements of each of these cells were individually followed with a trackball over a period of 90 min. Every 2 min, the x and y coordinates of the screen pointer were registered by the computer. Plotting these x and y data on the screen reconstructed the path of each cell. From the obtained migration data, velocity (excluding migration breaks) and migration distance were calculated for each cell and the mean for all 40 cells was taken.
Actin polymerization. Bone marrow cells were resuspended in PBS at a concentration of 5 x 107 cells/ml, preincubated for 5 min at 37°C, and exposed to IL-8 for various times. The cells were then fixed with 3.7% paraformaldehyde-PBS for 30 min at room temperature and rinsed in buffer containing 0.2% gelatin and 0.01% Triton X-100. Cells were then incubated for 45 min in the dark with 5 µg of fluorescein-phalloidin (Sigma)/ml diluted in PBS. After washing and resuspension in PBS, the fluorescence of gated Gr1-positive cells was measured by flow cytometry.
The intracellular F-actin distribution was visually verified by fluorescence microscopy. CD11b-positive cells purified from bone marrow (106) were resuspended in PBS, incubated with or without IL-8 (250 ng/ml) for 30 s at 37°C, loaded into the chamber of a slide (LAB-TEK; Nalge Nunc International) containing a solution of 5% paraformaldehyde-PBS, and sedimented for 30 min at room temperature. Supernatant was removed, and FITC-phalloidin staining was performed as described above. Cell preparations were mounted with fluorescent mounting medium (DAKO) and observed with a fluorescence microscope, and representative cells were photographed.
CD11b expression. Bone marrow cells were prewarmed in either PBS (without Ca2+ or Mg2+) or HBSS (with Ca2+ and Mg2+) plus 0.1% BSA for 5 min and then incubated with various concentrations of IL-8 for 5 min and 30 min at 37°C. After incubation, the expression levels of CD11b were determined by flow cytometry with anti-CD11b antibody.
Adhesion assay.
bEnd5 cells were plated at a density of 105 cells per well in 96-well plates 2 days prior to the test. Bone marrow cells (5 x 104 in 0.1 ml of HEPES) were added to confluent bEnd5 cells pretreated with tumor necrosis factor alpha (TNF-
) (100 ng/ml, 2 h) and allowed to adhere for 20 min at 37°C. IL-8 was added to a final concentration of 400 ng/ml where indicated. After incubation, plates were washed three times with HEPES buffer. Fifty microliters of 0.5% hexadecyltrimethylammonium bromide (Sigma) was added to each well for 30 min to lyse the cells. Myeloperoxidase activity was used as a marker enzyme to measure the number of leukocytes that adhered as described previously. Briefly, the optical density at 450 nm was measured after 15 min of incubation with 250 µl of 0.2 mg of dianisidine dihydrochloride (Sigma)/ml-0.4 M H2O2 in PBS.
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RESULTS |
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60% reduction on day 2 of culture). However, under inflammatory conditions, the number of S100A8-positive cells in the bone marrow of S100A9-deficient mice was similar to that of the wild-type mice (see Fig. 12B). To investigate the S100A8 expression in peripheral cells, flow cytometric analysis of blood cells (Fig. 2A) and immunostaining of the cytospin preparations of thioglycolate-elicited peritoneal PMNs and the cryosections of spleens (Fig. 3) from S100A9-deficient mice was conducted. These experiments as well as Western blot experiments (Fig. 2B) failed to detect S100A8, indicating that, in peripheral mature S100A9-null cells, the S100A8 protein is either absent or at least not detectable with our antibody. However, Northern blotting and RT-PCR analysis (Fig. 2C and D) showed the presence of the S100A8 mRNA transcripts in various organs from S100A9-deficient mice, suggesting that the strong down-regulation of S100A8 protein relies upon a posttranscriptional mechanism. TEM in vitro is altered. To investigate the migratory properties of deficient neutrophils quantitatively, we performed in vitro assays to determine the TEM of bone-marrow-derived cells (Fig. 6). During 2 h of incubation, 3% of wild-type cells spontaneously migrated across an unstimulated bEnd5 mono-layer. Surprisingly, the S100A9-deficient cells (13.5%) displayed a higher spontaneous migration activity in this assay. The percentage of cells that migrated was calculated by dividing the number of cells found in the lower well by the total number of cells added on top of the bEnd5 monolayer in the upper well.
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Chemokinesis of S100A9-deficient neutrophils in a 3-D collagen matrix.
To further characterize the migratory properties of S100A9-negative neutrophils, we measured various dynamic parameters (cell velocity, migration rate, and migration distance) within a 3-D collagen matrix in the absence or presence of additional external stimuli. The increased spontaneous activity of S100A9-deficient neutrophils observed in TEM could not be confirmed in the 3-D collagen matrix assay (Fig. 7). The migration distance and the number of simultaneously migrating cells, as well as the velocity of S100A9-/- and S100A9+/+ neutrophils, were similar. Stimulation of the cells with IL-8 was accompanied by significant changes in the dynamic parameters (Fig. 7). The migration rate of wild-type cells was slightly higher than that of the S100A9-deficient cells. This increase could be due to both the recruitment of previously nonmigrating cells and the increased duration of migration. Additionally, wild-type cells were able to cover greater distances than S100A9-deficient cells (70 and 50 µm, respectively). Cell velocity of wild-type peritoneal neutrophils was also higher than that of deficient neutrophils after stimulation with IL-8. The differences were significant according to statistical evaluation (P < 0.05).
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IL-8 may not be able to enhance adhesion of S100A9-deficient neutrophils to bEnd5 endothelial cells.
As reported earlier (3, 10, 24), IL-8 stimulates neutrophils to adhere to endothelial cells by an up-regulation of ß2 integrin CD11b surface expression. Therefore, we analyzed the adhesion of S100A9-deficient neutrophils to bEnd5 endothelial cells. No IL-8-dependent increase of adhesion of S100A9-deficient cells could be detected, whereas IL-8 stimulation of wild-type neutrophils enhanced adhesion to TNF--activated bEnd5 cells (Fig. 9). However, statistically, the difference seems to not be significant. The number of adherent cells did not differ when mutant and wild-type neutrophils were not stimulated with IL-8 (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The S100A9-deficient mice were viable and appear to be healthy. This is in sharp contrast to S100A8-deficient animals. The S100A8-deficient embryos were resorbed by day 9.5 in the uterus. This may indicate an unexpected function of S100A8 in early embryogenesis (22). S100A9 is obviously dispensable for this function. The energetically favored heterodimerization of S100A8 and S100A9 and their almost identical expression pattern leads to the assumption that the heterodimers are the biologically active molecules. The difference in the viability of both mouse models suggests that both molecules bear biological functions independent of heterodimerization.
Furthermore, the peripheral S100A9-null leukocytes lack detectable amounts of S100A8. Thus, even the lack of both S100A8 and S100A9 comprising nearly 40% of the cytosolic proteins in wild-type neutrophils does not affect the development of viable and mature neutrophils. Moreover, we could not detect an up-regulation of any other S100 protein (data not shown). However, it is important to note that S100A8 transcription is not altered in the S100A9-deficient mice and that residual S100A8 below the detection limit may be present in peripheral S100A9-deficient neutrophils.
S100A8 is present in bone marrow cells. Our observation that the percentage of S100A8-positive cells in the bone marrow of S100A9-deficient mice increased after inflammatory stimulation of granulopoiesis suggests that S100A8 protein is expressed in early differentiation stages and may become unstable in mature granulocytes.
We investigated the myelopoietic potential in S100A9-deficient mice. The S100A9-deficient animals displayed a normal population of myeloid cells in the bone marrow, as shown by immunocytochemistry. It has been speculated in an earlier report that both S100 proteins may be involved in the terminal differentiation of myeloid cells by inhibiting casein kinase (18). Our results indicate that the deficiency of S100A9 is dispensable for myelopoiesis and mobilization. However, the reduced number of IL-3-responsive cells and the increased number of G-CSF- and M-CSF-induced colonies derived from the S100A9-deficient bone marrow allow speculation that a potential defect in early myelopoiesis may be compensated by an unknown mechanism.
The most prominent property of neutrophils is their ability to invade inflamed tissue. S100A9 and S100A8 have both been described as molecules involved in adhesion (22, 25, 30) and directed migration (14). The unusually high F-actin content in S100A9-deficient neutrophils functionally links S100A9 to cytoskeletal regulation. Actin filaments are the primary cytoskeletal elements mediating migration. A functional actin cytoskeleton is essential for the coordinated expression and cell surface exposition of integrins. Integrins are linked to actin filaments by a number of actin-binding proteins, and the integrin-dependent cell spreading of PMNs requires actin polymerization (2). The deregulated microfilament system may cause the abnormal induction of the CD11b surface expression observed in S100A9-deficient neutrophils. Numerous reports associate various S100 proteins with the regulation of cytoskeletal dynamics (5, 26, 32). S100A1 and S100B are supposed to be involved in microtubule metabolism. S100A1 additionally inhibits phosphorylation of the actin-capping molecule MARCKS. S100A10 is proposed to regulate the activity of annexin II, including its association with cytoskeleton constituents and its F-actin-bundling activity (5). Moreover, in striking similarity to our analysis, a more organized microfilament formation has also been shown in C6 glioma cells in which the expression of S100B has been selectively inhibited by antisense technology (28).
It has been reported that S100A8- and S100A9-positive cells express more CD11b than S100A8- and S100A9-negative monocytes (6) and belong to the first infiltrating cells in inflammatory lesions. The finding that the actin filament system is highly polarized in S100A9-deficient cells correlates with a reduced ability to migrate upon a stimulus, as shown in the 3-D collagen matrix migration assay. The failure to up-regulate CD11b may result in the absence of IL-8-mediated adhesion. IL-8 and LTB4 (29) are known to up-regulate CD11b surface expression. The reduced ability of LTB4 to stimulate the TEM of S100A9-deficient cells suggests that LTB4 may also fail to enhance S100A9-deficient neutrophil adhesion. Consequently, both the disturbed microfilament system and the absence of chemoattractant-induced adhesion may be responsible for the strong attenuation of the chemotactic response, as observed with S100A9-deficient neutrophils in the TEM assay. The inability of S100A9-deficient cells to enhance CD11b surface expression upon stimulation weakens the potential of these cells to adhere and may thus explain the discrepancy between the diminished responsiveness to chemoattractants observed in the TEM assay and the less pronounced effect found in the collagen matrix assay. Furthermore, extracellular S100A9 is known to modulate the affinity of the CD11b integrin receptor (21) and may facilitate transmigration by contributing to the binding to endothelial heparan sulfate (25) and/or carboxylated proteoglycans (30), suggesting that reduced transmigration may additionally result from the lack of extracellular S100A9. However, applying two inflammatory models, we could not detect any significant difference in the leukocyte invasion in vivo by using S100A9-deficient mice. It is known that different inflammation models involve different inflammatory mediators and adhesion molecules. CD18-deficient mice unexpectedly show no inhibition of neutrophil emigration during sterile peritonitis by the intraperitoneal injection of thioglycolate and, on the contrary, even an increased emigration in other models (4, 17). Similarly, migration of PMNs in the thioglycolate peritonitis model is enhanced in mice lacking the tyrosine kinases Hck, FGr, and Lyn, known as members of the integrin-signaling pathway (16) as well as in CD11b-deficient mice (15). Obviously, there is more than one pathway orchestrating neutrophil emigration into inflamed tissue. The role of S100A9 and S100A8 may still prove to play a major role in in vivo inflammatory models other than those investigated here.
Most transduction pathways that induce cell migration involve components that regulate the intracellular calcium concentration. Calcium in turn acts on actin-associated proteins like MARCKS and gelsolin (29) and thereby regulates actin polymerization. The observation that firstly, the CD11b surface expression is reduced in calcium-free buffers in S100A9-deficient cells, that secondly, extracellular calcium restores normal CD11b surface expression, and that the strictly calcium-dependent, IL-8-mediated up-regulation of CD11b cannot be observed in S100A9-deficient neutrophils suggests that primarily the calcium homeostasis of S100A9-deficient cells is affected. Alterations in Ca2+ homeostasis have also been reported in mice deficient in calbindin, S100B, and calretinin, all calcium-binding EF-hand proteins expressed in the cells of the central nervous system (1, 27, 33). Further investigations are in process to define the role of the S100A9 molecule in neutrophil's calcium metabolism.
Using gene-targeting technology, we demonstrated that loss of the massively expressed S100A9 protein results in a viable and obviously healthy mouse. However, we observed a dramatic posttranscriptional down-regulation of S100A8 protein. On the cellular level, S100A9-deficient neutrophils exhibited a more organized microfilament system and a reduction of IL-8 mediated CD11b surface up-regulation, resulting in a diminished potential to respond to chemoattractant molecules in vitro. Nonetheless, we could not detect any abnormal leukocyte emigration that applied two inflammatory in vivo models, suggesting that S100A8 and S100A9 molecules may be dispensable, at least for the inflammatory models investigated here.
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ACKNOWLEDGMENTS |
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The excellent technical assistance of D. Wiesmann, B. Küter-Luks, K. Fischer, and H. Hater is acknowledged. Many thanks to A. Kondrashov for his help with blastocyst injections. We also thank Claus Kerkhoff for critical reading of the manuscript.
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FOOTNOTES |
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