In vitro generation of mature neutrophils from canine Lin− bone marrow cells

Veterinary Immunology and Immunopathology 107 (2005) 41–50 www.elsevier.com/locate/vetimm

In vitro generation of mature neutrophils from canine Lin bone marrow cells Leticia G. Leo´n a, Luciana K. Ostronoff a, Marı´a Luisa Fermı´n b, Cristina Fragı´o b, Elisabeth Kremmer c, Hans-Jochem Kolb d, Concepcio´n Tejero a,* a

Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain b Departamento de Medicina y Cirugı´a Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain c Institut fu¨r Immunologie, GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Marchioninstr 25, 81377 Mu¨nchen, Germany d Institut fu¨r Haematologie, GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Marchioninstr 25, 81377 Mu¨nchen, Germany Received 27 July 2004; received in revised form 23 December 2004; accepted 14 March 2005

Abstract The major goal of this work was to describe the in vitro generation of mature functional neutrophils derived from a canine enriched haematopoietic progenitor cell population. We have utilised lineage depletion by immunomagnetic selection to isolate a canine haematopoietic progenitor cell population. The physical, immunological, metabolical and morphological methodologies employed in this study have permitted us to isolate and define a cell population enriched in Rh-123low and CD34+ cells. Irradiated pre-established long-term bone marrow cultures (LTBMC) were utilised to determine the self-renewal ability of lineage negative (Lin ) cells, as well as their capacity to differentiate into mature functional neutrophils. The authors demonstrate for the first time that canine neutrophils derived from Lin cells are able to produce oxyradicals, express a specific neutrophil surface antigen, and contain gelatinase granules. These characteristics enable them to migrate through basement membranes to act as a first line defence mechanism. The fact that these cells are able to differentiate into functional mature cells, and give rise to long-term culture-initiating cells (LTC-IC) after 35 days of culture, allows the authors to assure that the isolated canine enriched haematopoietic cell population exhibit functional characteristics, associated with primitive haematopoietic cells. # 2005 Elsevier B.V. All rights reserved. Keywords: Canine haematopoiesis; Lin

population; In vitro differentiation; Neutrophil functionality

Abbreviations: BFU-E, burst forming unit-erythroid; cG-CSF, canine granulocyte-colony-stimulating factor; cSCF, canine stem cell factor; CFU-GM, colony forming unit-granulocyte-macrophage; DHR, dihydrorhodamine 123; FACS, fluorescent-activated cell sorting; hEPO, human erythropoietin; hGM-CSF, human granulocyte/macrophage-colony stimulating factor; Lin , lineage negative; Lin+, lineage positive; LTBMC, long-term bone marrow culture; LTC-IC, long-term culture-initiating cells; MMP, metalloproteinase; MWM, molecular weight markers; PE, phycoerythrin; Rh-123, rhodamine 123 * Corresponding author. Tel.: +34 913 943899; fax: +34 913 943909. E-mail address: [email protected] (C. Tejero). 0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.03.014

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1. Introduction Haematopoiesis depends on the continuous proliferation, expansion and differentiation of a small compartment of primitive cells, which comprises haematopoietic stem cells (HSC) and their progeny. This population is a minor component of the total number of marrow cells. The evident clinical relevance of HSC in bone marrow transplantation, and the possibility of using gene transfer methods for correcting genetic diseases have already been implemented. The most primitive category of HSC is usually reserved for those cells that are the least differentiated, and have the highest replicative potential; these were thought to be intrinsic characteristics of metabolically inactive cells. It has been shown that the uptake of rhodamine 123 (Rh-123) is minimal in non-cycling cells (Johnson et al., 1980; Ploemacher and Brons, 1988; Kim et al., 1998). The use of Rh-123 dye efflux, in combination with phenotypic markers, might represent a great advantage to select haematopoietic progenitor cells (HPC) (Udomsakdi et al., 1991; Leemhuis et al., 1996; Uchida et al., 1996; Liu and Verfaillie, 2002). The repopulating potential of HSC can be defined as a process in which an input of cells proliferate and differentiate to give physiological mature cells, of specific haematopoietic lineages. This process can be assessed in vivo by allogeneic bone marrow transplantation in dogs (Kolb et al., 1997), and xenogeneic transplants, using immune-compromised animals (Barquinero et al., 2000; Niemeyer et al., 2001). As an alternative to in vivo experiments, in vitro assays have been used to measure the activity of HSC, including long-term culture-initiating cells (LTC-IC) assays (Sutherland et al., 1990; Punzel et al., 1999; Forraz et al., 2004). In this context, it is important to ascertain that homing, differentiation and maturation processes of HSC, in the course of the in vitro system, develop a functional progeny. Mature leukocytes such as neutrophil granulocytes respond to bacterial infections by secreting substances such as cytokines, oxyradicals, reactive nitrogen species, and enzymes (Witko-Sarsat et al., 2000). The ontogeny of myeloid cells involves sequential steps in which the acquisitions of specific granules occur. Specifically, gelatinase granules contain metalloproteinases (MMP) that enable neutrophils to cross

the extracellular matrix barriers to reach their target tissue at the inflammation sites, and act as a first line defence mechanism (Goetzl et al., 1996). In fact, biogenesis of gelatinase granules is considered a marker of terminal neutrophil differentiation (Borregaard et al., 1995). Canine models have proved to be predictive of clinical findings in human bone marrow transplantation; consequently, the utilisation of dogs can be an excellent tool to improve therapeutic purposes (Ladiges et al., 1990; Kolb et al., 1997). For this reason, many efforts have been made to characterise the canine haematopoietic system. However, less is known regarding characterisation and isolation of canine haematopoietic progenitor cells, while canine CD34 has been cloned (McSweeney et al., 1996, 1998). Herein, we used a combination of several approaches to in vitro generate a mature functional neutrophils obtained from a canine enriched haematopoietic cell population. The expression of a specific antigen as well as the acquisition of MMP and the ability to produce oxyradicals, could serve as sensitive markers for monitoring neutrophil differentiation and maturation.

2. Materials and methods 2.1. Animals Six healthy spayed female Beagles between 2 and 3 years of age were used as marrow and blood donors. Bone marrow specimens were obtained from the iliac crest and wing of the ilium, using a sterile technique, under intravenous anaesthesia with medetomidine (18 mg/kg) and diazepam (0.4 mg/kg). Blood samples were obtained in heparin–lithium, by clean venipuncture of the jugular vein. The dogs used in this study were treated according to the European Community (EC) Council Directive (86/609, 1986) for the care of experimental animals. 2.2. MNC isolation and magnetic cell sorting Bone marrow was diluted in PBS and mononuclear cells (MNC) were obtained by density-gradient centrifugation onto Ficoll-Paque PlusTM (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Lineage

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depletion and positive selection of CD34+ cells was realized using magnetic cell sorting methodology according to the manufacturer’s instructions (Miltenyi Biotech, Galdabach, Germany). For depletion procedure, MNC were labelled with lineage-specific antibodies direct against canine antigens: Dog 13 (anti-Thy-1), Dog 15 (anti-granulocytes, monocytes), Dog 17 (anti-CD5), Dog 22 (anti-IgG), Dog 26 (antiMHCII) (Cobbold and Metcalfe, 1994; Neuner et al., 1997). The antibodies were used as cell culture supernatants in a dilution of 1:10, which has been found a saturating concentration for all antibodies. After MACS procedure, a median of 16.3  0.5% (n = 15) of MNC were recovered in the Lin fraction. Purities were routinely greater than 94%, for both positive and negative fractions. Positive selection of CD34+ cells was performed with 1H6 monoclonal antibody (Becton Dickinson, San Jose, USA) at 40 mg/ ml (McSweeney et al., 1998). 2.3. Long-term bone marrow culture assay Canine long-term bone marrow cultures (LTBMCs) were established as previously reported (Fermı´n et al., 2004). Cultures were fed weekly by removing the total amount of medium and replacing it with an equal volume of fresh medium. Fourteen-day-old cultures were irradiated with 15Gy (X-ray, 86.61 rad/min 17 min 20 s) to abolish endogenous haematopoiesis, and recharged with Lin cells (106 cells/flask). Cultures were fed weekly as described above. One week later, cobblestone areas were observed indicating the adhesion of progenitor cells to the stroma layer. On days 7 and 14 after culture irradiation, generated non-adherent cells were harvested to determine the number of mature neutrophils and the expression of surface antigens (Dog 15) by flow cytometry. Gelatinase activity was assayed on days 7, 14 and 21 following irradiation. Respiratory burst of neutrophils was measured on day 7. Between days 28 and 35 after Lin input, LTBMC adherent cells were assayed for the presence of LTC-IC.

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(5  104 cells/ml) were assayed for CFU in IMDM containing 20% pooled allogeneic dog serum (DS), 1.4 mM glutamine and 0.33% (w/v) agar (Difco laboratories, Detroit, USA). These cells were stimulated with 100 ng/ml human granulocyte/macrophagecolony-stimulating factor (Biosource International, Camarillo, USA) and 1 U/ml human erythropoietin (Roche Diagnostics, Indianapolis, USA). Rh-123low cells (2.8  104 cells/ml) were assayed in the same medium but stimulated with 100 ng/ml canine stem cell factor, 100 ng/ml canine granulocyte-colonystimulating factor and 1 U/ml human erythropoietin (Roche Diagnostics, Indianapolis, USA) (canine growth factors kindly provided by Amgen, Thousands Oaks, CA, USA). Cultures were plated in 3 wells of 24well tissue culture plates and incubated in a humidified atmosphere at 38 8C and 5% CO2. Colonies (>50 cells) were scored using an inverted microscope after 11–14 and 19 days for MNC and Lin and Rh-123low samples, respectively. Colonies were classified as follows: BFUE, erythroid colonies of hemoglobinized cells grouped in one or several clusters, and CFU-GM, colonies containing both granulocytes and macrophages. The data are expressed as CFU/105 plated cells. 2.5. LTC-IC assays The ability of Lin population to generate CFU after culturing on competent stromal layer was determined as previously described with modifications (McSweeney et al., 1998). Irradiated LTBMCs were recharged with Lin cells as outlined under LTBMC assay. After 28–35 days, the adherent layer was harvested by trypsinization and assayed in agar culture system for the presence of CFU, as described above. The assays were performed with 105 cells/ml, and 100 ng/ml canine stem cell factor, 100 ng/ml canine granulocyte-colony-stimulating factor and 1 U/ml human erythropoietin were added to stimulate colony formation. Colonies (>50 cells) were scored after 16 days. The number of LTC-IC was defined as the number of colonies grown in these cultures.

2.4. Colony-forming assay 2.6. Flow cytometry and FACS analysis CFU assays were performed using a modification of previously described procedures (Coutinho et al., 1993). MNC (7.5  104 cells/ml) and Lin cells

Bone marrow MNC and Lin cells were stained by incubation with 100 ng/ml (final concentration)

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Rh-123 (Sigma Chemical Co., St. Louis, USA) for 20 min at 37 8C. After one wash, the cells were resuspended in PBS and incubated (20 min at 37 8C) to permit Rh-123 efflux. Cells were washed again and finally resuspended in PBS. Two Lin /Rh-123 cell populations were isolated by FACS on the basis of Rh123 retention, Lin /Rh-123low (21.2  3%, n = 4) and Lin /Rh-123high (46.2  8.8%, n = 4). The purity of Lin /Rh-123low and Lin /Rh-123high populations was 78  5% (n = 3) and 83  3% (n = 3), respectively. Cytospin preparations of this population stained with May-Gru¨ nwald-Giemsa revealed that 50% of the nucleated cells were myelo-lymphoblasts without defined granules. The expression of specific granulocyte surface antigens on LTBMC non-adherent cells was determined using Dog 15 monoclonal antibody. Peripheral blood and LTBMC non-adherent cells were incubated with Dog 15 for 30 min at 4 8C. The cells were washed once and incubated with phycoerythrin (PE)-conjugated goat–anti-rat antibodies (Serotec, Oxford, UK) for 15 min at 4 8C. Once washed, the cells were resuspended in PBS for analysis. Dead cells were excluded from these analyses by staining with 1 mg/ml propidium iodide (Sigma Chemical Co., St. Louis, USA). Flow cytometry was performed on a FACScan (Becton Dickinson, San Jose, USA) and cells were sorted on a Coulter Epics Elite (EPS). The data obtained were analysed using WinMDI 2.8 free software. 2.7. Gelatin zymography Gelatin zymography was carried out as recently reported in detail (Fermı´n et al., 2004). Briefly, Lin cells and peripheral blood and culture grown neutrophils, obtained by density-gradient centrifugation onto Ficoll-PaqueTM (Amersham Pharmacia Biotech AB, Uppsala, Sweden), were resuspended in nonreducing SDS sample buffer. Samples were fractionated in 10% acrylamide gels containing 0.1% pork skin gelatin (Sigma Chemical Co., St. Louis, USA). After the run, gels were incubated at room temperature with 2.5% Triton X-100 for 1 h, with one change at 30 min. Then the gels were washed once with the digestion buffer (50 mM Tris, pH 7.5; 200 mM NaCl; 5 mM CaCl2; 1 mM ZnCl2)

and incubated in the same buffer for 18 h at 37 8C. In order to inhibit metalloproteinase activity, 5 mM EDTA (final concentration) was added to the incubation buffer and the gels were incubated as described (Ries et al., 1999). The zymograms were stained with 0.5% Coomassie blue R-250 in 30% methanol, 10% acetic acid. Clear zones of gelatin lysis against a blue background indicated enzyme activity. 2.8. Dihydrorhodamine flow cytometry assay Neutrophil activation by PMA was assessed by flow cytometry based on the oxidation of dihydrorhodamine 123 (DHR) to its fluorescent derivative, Rh-123 (Lieberman et al., 1996; Richardson et al., 1998). LTBMC non-adherent cells were separated by density centrifugation onto Ficoll-PaqueTM (Amershan Pharmacia Biotech AB, Uppsala, Sweden). Neutrophils were collected, washed once with 0.9% NaCl containing 20% FCS and resuspended in PBS. For each experimental point, three 100 ml samples (106 cells) were taken. These were used as stimulated (PMA + DHR), resting (DHR) and reagent blank (only cells) test. After the addition of 25 ml PMA (3 mg/ml) (Sigma Chemical Co., St. Louis, USA), all tubes were incubated at 37 8C for 15 min. Then 25 ml DHR (30 mg/ml) (Sigma Chemical Co., St. Louis, USA) was added. The samples were incubated for additional 5 min. After centrifugation, the cells were resuspended in 600 ml of 1% paraformaldehyde in PBS. Dead cells were excluded from these analyses by staining with 1 mg/ml propidium iodide (Sigma Chemical Co., St. Louis, USA). 2.9. Morphological studies Mononuclear, Lin , as well as non-adherent cells obtained from LTBMC were prepared for cytospin and examined on slide preparations stained with MayGru¨ nwald-Giemsa stain. Approximately, 500–1000 cells per slide were scored. 2.10. Statistics Data are presented as the mean  standard error of the mean (S.E.M.).

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3. Results

3.2. Proliferation and differentiation of Lin into mature neutrophils

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cells

3.1. Lineage depletion and rhodamine staining The Lin population obtained by inmunomagnetic selection gave a 6  0.3-fold enrichment of BFU-E and a 4.3  0.7-fold enrichment of GM-CFU, when comparing to MNC. The depletion procedure greatly increased the number of myeloid and erythroid precursors (Fig. 1), in parallel with CFUs enrichments. In addition, the percentage of CD34+ cells raised from 3.1  0.7% (n = 3) in MNC to 6.1  1.6% (n = 5) in lineage depleted cells. Rhodamine staining confirmed the enrichment in the Rh-123low population. Depletion procedure highly increased the percentage of Rh123low cells in Lin population (21.2  3%) over MNC (11.3  1.6%). Moreover, Lin /Rh-123low population generated 107 CFU/105 cells when incubated with cSCF, cG-CSF and hEpo. Colonies were scored on day 19 and their morphology was small and tight (Fig. 2).

We evaluated the potential of Lin cells to adhere to the stroma, grow and differentiate into mature neutrophils using irradiated pre-established LTBMC. Every 7 days after Lin cells input, the non-adherent cell population produced in the culture was analysed for the expression of a specific granulocyte antigen, the acquisition of gelatinase granules and the ability to produce superoxide anion. One week after the Lin input, a normal stroma layer with typical cobblestone areas was established and observed during the life span of the culture (data not shown). The adherent cells were harvested 4–5 weeks after culture irradiation and were assayed for LTC-IC. The content of LTC-IC was 341  101/105 plated cells (n = 3) (Fig. 2). The expression of a granulocyte antigen in culture grown neutrophils was determined by flow cytometry, using a specific anti-canine antibody. Fig. 3 shows

Fig. 1. Enrichment in myeloid and erythroid precursors after depletion. Cytospin preparations of bone marrow MNC and lineage-depleted cells were stained with May-Gru¨ nwald-Giemsa. Microphotographs of mononuclear (A) and Lin (B) cells are shown. Cells were visualised by light microscopy and photographed at 1000 (MNC) and 600 (Lin ) original magnification. (M) and (E) indicate myeloid and erythroid precursors, respectively (bar, 0.5 cm). (C) Differential counts of canine nucleated cells before and after depletion.

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Fig. 2. Sorted Lin /Rh-123low population and adherent cells from days 28–35 after the input of Lin cells to pre-established irradiated LTBMC were assayed for the presence of CFU. Colonies were visualised by inverted light microscopy and photographed at 250 original magnification.

representative dot plots of peripheral blood and culture grown neutrophils labelled with monoclonal antibody Dog 15. Neutrophils represented 63.1  5.3% (n = 3) and 35.9% (n = 2) of the non-adherent cell population 7 and 14 days after adding Lin cells to the irradiated cultures, respectively. In parallel, the non-adherent cell population was examined in cytospin preparations. Most of the cells in this population were segmented neutrophils (58.1  1.8%, n = 4 and

48.9  10.7%, n = 3, for day 7 and day 14 nonadherent cells, respectively). Moreover, the expression of gelatinase in these cells was analysed to demonstrate neutrophil differentiation. Peripheral blood and culture grown neutrophils, purified by centrifugation onto FicollPaqueTM, were fractionated by SDS-polyacrylamide gel electrophoresis containing 0.1% gelatin, and submitted to zymography. Fig. 4 reveals the presence of two gelatinolytic enzyme activities in peripheral blood neutrophils, migrating at 92 and 72 KDD. Culture grown neutrophils derived from Lin cells displayed the gelatinolytic activity at 92 KDD. Furthermore, Lin cells did not contain any kind of enzyme activity. Addition of EDTA to the enzyme activation buffer, during the development of the zymography, inhibited the two activities (data not shown), indicating that they both belong to the metalloproteinase subclass of enzymes. The 92 KDD activity coincides in molecular weight with gelatinase B, a matrix metalloproteinase produced by neutrophil granulocytes (Makowski and Ramsby, 1996; Pugin et al., 1999). However, the presence of a 72 KDD gelatinase in neutrophils has not been described. It should be mentioned that the appearance of a 72 KDD activity in peripheral blood neutrophils depends on the number of cells analysed, which can be observed from 4000 neutrophils onwards (data not shown). To evaluate neutrophil respiratory burst, a flow cytometric assay, based on the intracellular oxidation of the nonfluorescent compound DHR-123 to its fluorescent derivative Rh-123, has been utilised (Richardson et al., 1998). Fig. 5 shows the physiological ability of these cells to produce oxyradicals when activated with PMA. Both peripheral blood and culture grown neutrophils developed green fluorescence as a result of DHR oxidation by hydrogen peroxide generated upon activation of the NADPH oxidase complex. The Rh-123 fluorescence displayed by LTBMC and peripheral blood neutrophils were very similar, varying between 69.9  3.0% (n = 3) and 84.1  3.3% (n = 3), respectively.

4. Discussion Our main objective was to describe the ability of a canine Lin population to in vitro generate mature

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Fig. 3. Representative dot plots of Dog 15 analysis in canine neutrophils. (A) Peripheral blood, (B) and (C) neutrophils harvested on days 7 and 14 after the input of Lin cells to pre-established irradiated LTBMC, respectively.

functional neutrophils. The dog turns out to be an excellent model for developing novel strategies in human bone marrow transplantation and gene therapy (Ladiges et al., 1990; Storb, 2003). However, information related to canine haematopoietic progenitor cells is still limited. In the present study, lineage depletion has been used in order to purify and define a population with characteristics of primitive haematopoietic cells. Data concerned to CFU assays, morphological studies and Rho-123 staining allows us to assert that the canine enriched haematopoietic cell population exhibits characteristics, associated with primitive haematopoietic cells. These results are in agreement with those described by Niemeyer et al. (2001) for the lineage depletion of canine bone marrow cells. We want to put emphasis on the fact that the Lin /Rh123low population, obtained by us, was able to produce CFU in soft agar after 19 days (Fig. 2). Other authors,

using depletion procedures, also reported increases in blast cell morphology after selection of a human CD34+ cell enriched population (Flores-Guzma´ n et al., 2002). It is important to guarantee that transplantable HSC are able to home, grow and differentiate, giving rise to mature functional cells. In this study, we have utilised an in vitro system to authenticate that the Lin cell population displays properties associated with haematopoietic primitive populations. In our context, we assume in vitro homing as the capacity of the progenitor cells to adhere to stroma layers (Frimberger et al., 2001). This is supported by weekly episodes of neutrophil production and the appearance and persistence of cobblestone areas during the life span of the cultures. In addition, the LTC-IC assay detected the repopulating ability of Lin progenitor cells by means of the growth of myeloid clonogenic cells after 4–5 weeks of Lin input (Fig. 2).

Fig. 4. Peripheral blood neutrophils (PB), Lin , and LTBMC neutrophils from days 7, 14 and 21 after the input of Lin cells were assayed for gelatin zymography on 10% SDS-polyacrylamide gel containing 0.1% gelatin. Approximately 5000 cells were loaded. The percentage of neutrophils was 90% for PB, and from 64 to 80% for culture grown neutrophils. Molecular weight markers (MWM) are indicated.

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Fig. 5. Respiratory burst of neutrophils by DHR assay. Representative dot plots of rhodamine fluorescence of stimulated PB (A) and LTBMC neutrophils from day 7 after the input of Lin cells (B) are shown.

In order to verify the lineage potentiality of the isolated cells, we have chosen the study of neutrophils during their differentiation and maturation processes. Normal functionality of neutrophils will protect the host against pyogenic infections acting as a first line defence mechanism. Neutrophil surface molecules undergo several changes during neutrophilic maturation to accommodate the cell’s function, and are responsible for sensing changes in the surrounding environment, acting as biological sensors. In normal human marrow, CD15 antigen was weakly expressed on blast cells and strongly expressed on more mature granulocytic cells (Terstappen et al., 1990). Canine Dog 15 recognises about 80% of peripheral blood and bone marrow granulocytes (Neuner et al., 1997). In our study, mature neutrophils derived from cultured Lin populations were labelled by Dog 15 antibody in a similar proportion as neutrophils from peripheral blood. Equally, their ability to produce oxyradicals, oxidising DHR-123 to Rh-123 is in the same range as those found in neutrophils from peripheral blood. Previous studies have shown how all components of the NADPH oxidase are expressed after myelocyte stage during neutrophil differentiation (Babior, 1999; Hua et al., 2000). Finally, as a conclusive evidence of the granulocytic line maturation process, we have analysed the expression of MMP-9 in neutrophils. Exocytosis of gelatinase from gelatinase granules is essential for migration of neutrophils through basement membranes (Delclaux et al., 1996). Gelatinase is

synthesised mainly in band and segmented cells (Borregaard et al., 1995). The gelatin zymogram shows a digested zone at 92 KDD molecular weight for mature neutrophils from peripheral blood, and those harvested from cultures. As we expected, no gelatinolytic activity was found in Lin cells, indicating the immature stage of cells belonging to Lin population. Yokota et al. (2001) has reported a 92 KDD matrix metalloproteinase-9 in canine mammary carcinoma. The physiological properties analysed in the neutrophils generated from Lin cells guarantee their capacity to act as members of the body defence system. The physical, immunological, metabolical and morphological methodologies employed in this study have permitted the isolation of a cell population enriched in Rh-123low and CD34+ cells able to in vitro differentiate into mature functional neutrophils. The findings of the present study provide an excellent model system for studying neutrophil development, maturation and function in transplantable precursor cells.

Acknowledgements We are very grateful to Paula Cerdeira, Ana Angulo and Felisa Garcı´a for their technical assistance. The authors wish to thank Antonio Castillo and Daniel Heys for English review of the manuscript. We are also very grateful to Amgen, for supplying the canine cytokines used in this study. This work was supported

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