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From the Cardiovascular Research Group, Division of Clinical Sciences North, University of Sheffield, Sheffield, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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70% of neutrophils from whole
blood. Flow cytometric analysis shows little difference in surface
L-selectin and CD18 expression on isolated neutrophils compared with
neutrophils in whole blood, indicating that neutrophils
are minimally activated bythe isolation process. Stimulation with
phorbol 12-myristate 13-acetate (PMA) reduced L-selectin
andincreased CD18 expression. Isolated neutrophilsmigrate
under agarose in response to fMLP, and fluorescently labeled
neutrophils transfused into recipient mice interact with postcapillary
venules in a manner comparable to endogenous leukocytes. These findings
show that neutrophils isolated using this method can be used for
inflammatory studies in vitro and in
vivo.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods currently available for the isolation of mouse neutrophils rely either on differences in the buoyancy of mouse blood cells for density gradient centrifugation16,17 or require a neutrophil-specific migratory stimulus to be administered in vivo into a body cavity.18,19 The former method does not permit reproducibly pure populations of neutrophils to be isolated, as differences in buoyancy between mouse leukocytes are not sufficient for easy separation. Hematological variation between different mouse strains further limits this technique. Using the latter method, neutrophils migrate into a cavity in response to an inflammatory stimulus to be isolated. Although this method elicits highly pure neutrophil populations, migrated neutrophils display a phenotype that is dramatically different compared to quiescent neutrophils, for example with regard to their internal cytoskeletal arrangement and surface expression of adhesion molecules and receptors.20-27 Results obtained using pre–migrated cells in models of inflammation may not be predictive because the neutrophils have already engaged in an inflammatory response.
In the present work we describe a novel method for the isolation of >95% pure populations of unactivated neutrophils from whole blood using negative immunomagnetic separation.28 Preliminary data shows that these isolated neutrophils are capable of both migration in response to chemokines in vitro and also rolling in postcapillary venules in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dextran (T500) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Bovine serum albumin, PMA, fMLP, and fluorescein isothiocyanate (FITC)-labeled rabbit anti-rat IgG were purchased from Sigma (Poole, UK). Rat anti-mouse antibodies to CD2 (RM2–5), CD5 (53-7.3) and CD45R (RA3-6B2), phycoerythrin (PE)-labeled rat anti-mouse Ly-6G (RB6-8C5), PE-labeled rat anti-mouse L-selectin (CD62L; MEL-14), CellWash and PharmLyse were purchased from PharMingen (Oxford, UK). Rat anti-mouse ICAM-1 (YN1/1) was a gift from Dr. C. Wegner (Abbott Laboratories, Abbott Park, IL). Rat anti-mouse F4/80 antigen (CI:A3-1), FITC-labeled rat anti-mouse CD18 (C71/16), PE-labeled rat IgG2a isotype-negative control, and FITC-labeled rat IgG2a isotype-negative control were purchased from Serotec (Kidlington, UK). Goat anti-rat IgG microbeads were obtained from Miltenyi Biotech (Bisley, UK). Falcon 3001 tissue culture dishes were purchased from Becton Dickinson (Oxford, UK). Agarose (LE analytical grade) was purchased from Promega (Southampton, UK). RPMI-1640 and Hanks’ balanced salt solution were purchased from Life Technologies Ltd. (Paisley, UK). Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) was purchased from Molecular Probes (Leiden, Netherlands).
Immunomagnetic Cell Separation
Mice (25 to 30 g) were anesthetized with an intraperitoneal injection of ketamine (Ketaset, 125 mg/kg; Willows Francis Veterinary, Crawley, UK) and acepromazine (2.75 mg/kg; C-Vet Veterinary Products, Lancashire, UK). Heparin (50 U) was also administered intraperitoneally with anesthetic. Blood (1 ml) was collected by cardiac puncture using a heparinized syringe and transferred into dextran (3 ml, 1.25% w/v in saline). Tubes were then filled to a total of 10 ml with dextran solution and inverted. Erythrocytes were allowed to sediment for 30 minutes at room temperature and the leukocyte-rich supernatant collected. Cells were washed with buffer [sterile-filtered phosphate-buffered saline (PBS) without cations containing 0.5% w/v low-endotoxin bovine serum albumin, pH 7.4, 4°C] and a total leukocyte count was performed. A leukocyte differential count for the strain of donor mouse used was also performed. Antibodies to cell surface markers were selected based on published data29,30 to specifically label non–neutrophil cell types. In murine peripheral blood these are lymphocytes and monocytes. Anti-CD2, anti-CD5, and anti-CD45R were chosen to specifically label lymphocytes, anti-F4/80 antigen to specifically label monocytes, and anti-ICAM-1 as a pan-lymphocyte/monocyte antibody. Flow cytometry was used to determine saturating doses of antibody that specifically bound non–neutrophil cell types (see below). Whole–blood was then incubated at 4°C for 30 minutes with an antibody cocktail at concentrations based on the total number of lymphocytes and monocytes in the sample. Final antibody doses were as follows: anti-CD2 (1.5 µg/106 lymphocytes), anti-CD5 (2 µg/106 lymphocytes), anti-CD45R (10 µg/106 lymphocytes), anti-F4/80 antigen (2 µg/106 monocytes), and anti-ICAM-1 (0.6 µg/106 leukocytes). After removal of excess antibody by addition of 8 ml of buffer and centrifugation (6 minutes, 300 x g, 4°C), cells were resuspended in PBS (80 µl) and incubated with goat anti-rat IgG MicroBeads (20 µl/107 cells) at 4°C for 15 minutes. A chilled BS separation column was connected to a VarioMACS magnet (Miltenyi Biotech, Bisley, UK) and prepared with cold sterile water and buffer according to manufacturer’s instructions. The leukocyte/microbead mixture was then added to the column and the neutrophil-rich effluent collected. The unwanted cells, previously labeled with magnetic beads, were retained within the metallic matrix of the column. The neutrophil-rich effluent was then centrifuged (6 minutes, 300 x g, 4°C), the supernatant discarded and residual erythrocytes removed by hypotonic lysis. Hypotonic lysis was performed by the addition of 7 ml of 0.2% NaCl solution, gently inverted x10, followed by hypertonic rescue of neutrophils with an equivolume of 1.6% NaCl solution supplemented with 0.1% glucose and inverted once. Neutrophils were washed of erythrocyte debris and resuspended in PBS. Total cell counts were performed using a hemocytometer and differential cell counts were made using cytospins of a sample (100 µl) of the final neutrophil-rich cell suspension stained with Diff-Quick rapid staining set (BDH, Poole, UK) and neutrophils identified by their multilobular nuclei. From these data the yield and purity of the preparation was established.
Determination of Neutrophil Purity
We have used two methods to determine neutrophil purity. First, we used the traditional method of differential counts of cytospin as described above and, in addition, we have performed flow cytometric analysis of murine leukocytes before and after negative immunomagnetic separation. For flow cytometry, whole-blood leukocytes were identified on a dot plot by forward- and side-scatter characteristics. The neutrophil subpopulation was separately identified using PE-conjugated granulocyte-specific antibody RB6-8CS as previously described.13 Statistical analysis of the resulting neutrophil gate revealed the number of neutrophils as a percentage of the acquired leukocyte events. Using the same acquisition parameters, the above protocol was repeated using cell suspensions after negative immunomagnetic separation to determine the increase in purity of neutrophil isolates.
Flow Cytometry for Determination of Antibody Doses
Animals were anesthetized as described above and whole-blood was collected into ethylenediaminetetraacetic acid-coated tubes (Becton Dickinson, Oxford, UK). To determine leukocyte concentration, 10 µl blood samples were diluted 1:10 with 0.9% acetic acid and counted on a hemocytometer. Total lymphocyte/monocyte number per blood sample was then determined using differential counts of whole-blood smears. Blood samples (100 µl) were dispensed into flow cytometry tubes followed by different doses (µg/106 cells) of each rat anti-mouse primary antibody directed against CD2, CD5, CD45R, F4/80 antigen, or ICAM-1 to appropriate tubes. All tubes were incubated at 4°C for 30 minutes. Volumes were then expanded with 2 ml of CellWash followed by centrifugation at 300 x g for 6 minutes at 4°C. FITC-labeled secondary antibody (anti-rat IgG) was added to primary antibody-labeled cells. Secondary antibody was also added to unlabeled blood samples as a control of nonspecific binding. PE-labeled antibody against mouse Ly6G was added to whole-blood samples to specifically identify the granulocyte population. All tubes were incubated at 4°C for 30 minutes. To remove erythrocytes, 2 ml of PharmLyse was added to each tube and incubated for 15 minutes at room temperature, protected from light. All tubes were centrifuged as before, cell pellets were resuspended in 2 ml of CellWash, and centrifugation repeated. Cell pellets were finally resuspended in 500 µl of PBS for flow cytometric analysis using a FacScan cytometer (Becton Dickinson, Oxford, UK). Autofluorescence and secondary antibody control fluorescence were recorded on fluorescence intensity histograms. FITC fluorescence of the entire blood sample was obtained. Incremental shifts of the fluorescence intensity peak to the right indicated greater secondary antibody binding, and therefore greater primary antibody binding. To assess if neutrophils were labeled with primary antibody, the anti-Ly6G gate was used to determine any significant change in fluorescence intensity of the neutrophil population.
Viability and Activation Status of Isolated Neutrophils
Viability of isolated neutrophils was assessed by the exclusion of 0.1% trypan blue dye.31 The activation status of isolated neutrophils was determined by flow cytometric analysis of cell surface expression of L-selectin and CD18. Levels of these activation markers on isolated neutrophils were compared with levels on neutrophils in whole-blood, and on PMA-stimulated isolated neutrophils. Whole-blood neutrophils and isolated neutrophils were counted as above; isolated neutrophils were resuspended in PBS at a density of 1 x 106 cells/ml. Samples (100 µl) of whole-blood or isolated neutrophils were dispensed into flow cytometry tubes. As a positive control, a series of tubes containing isolated neutrophils were preincubated for 10 minutes at 37°C in the presence of PMA (10-7 mol/L). PE-conjugated anti-L-selectin, FITC-conjugated anti-CD18 or isotype control antibodies were then administered to appropriate tubes and incubated at 4°C for 30 minutes. To remove erythrocytes from whole-blood, 2 ml of PharmLyse was added to each tube and incubated for 15 minutes at room temperature and protected from light. All tubes were then centrifuged at 300 x g for 6 minutes at 4°C and supernatant removed. Cell pellets were resuspended in 2 ml of CellWash and centrifugation repeated. Cells were finally resuspended in 500 µl of PBS and analyzed using a FacScan cytometer.
In Vitro Neutrophil Chemotaxis
To determine whether isolated neutrophils could respond to inflammatory stimulation in vitro we observed the chemotactic response of isolated neutrophils in a previously described under-agarose assay.32 Briefly, 3 ml of agarose solution (50:50 Hanks’ balanced salt solution/RPMI-1640, 0.6% agarose) was poured into Falcon 3001 tissue culture dishes. Various concentrations of agarose were assessed to select conditions for optimal murine neutrophil migration. After the gels had solidified, two wells (diameter, 3 mm) were cut 2.2 mm apart on each plate. Isolated neutrophils (1 x 105 in 10 µl) were placed in the cell well and either fMLP (3 to 300 pmol in 10 µl of RPMI-1640 and 0.5% w/v bovine serum albumin) or control medium placed in the chemoattractant well. All plates were incubated for 2.5 hours in a 5% CO2 incubator at 37°C during which time neutrophil migration occurred. Cells were fixed to plates (4 ml methanol for 30 minutes followed by 4 ml 37% formaldehyde for 30 minutes) and, after removal of gels, stained with methylene blue. Images of plates were digitized [Nikon Eclipse E600 microscope (Nikon UK Ltd., Surrey, UK), RGB color digital TV camera (Basler Vision Technologies, Ahrensberg, Germany)] and distances of cell migration measured using image analysis software (Lucia DI; Nikon UK Ltd., Surrey, UK). Maximum migration distance (average of 20 furthest cells) from the edge of the cell well in the opposite direction to the chemoattractant source was subtracted from maximum migration distance in the direction of the chemoattractant to give a measure of directed migration.
In Vivo Neutrophil Rolling
The rolling of isolated neutrophils in vivo was analyzed by observation of the murine cremaster microcirculationusing intravital microscopy. Mice (25 to 30 g) were anesthetized with an intraperitoneal injection of ketaminehydrochloride (100 mg/kg) after a premedication of sodium pentobarbitone (Sagatal, 30 mg/kg; Rhône Mérieux Ltd., Essex, UK) and atropine sulfate (0.1 mg/kg; Pheonix Pharmaceuticals, Gloucester, UK). The trachea and carotid artery were cannulated with polythene tubing to aid respiration and permit administration of fluorescent-isolated neutrophils, respectively. The testis was exposed by a small scrotal incision and the cremaster muscle exteriorized and pinned over a glass coverslip. The cremaster tissue was superfused with thermocontrolled (37°C) bicarbonate-buffered saline equilibrated with CO2 in N2. Intravital microscopy was performed as previously described33 using a Nikon E600-FN microscope fitted with an epifluorescence attachment and immersion objectives. Isolated neutrophils were fluorescently labeled by incubation with CFDA-SE for 15 minutes at room temperature as previously described.34 Fluorescent neutrophils were injected (0.5 x 106 cells/200 µl) via the carotid artery and leukocyte-endothelial cell interactions in postcapillary venules within the cremaster were captured using a charge-coupled device camera (DC-330; DAGE MTI Inc., Michigan City, IN) and recorded onto sVHS video cassettes (AG-4700; Panasonic, Brackneu, UK). Image analysis was performed using NIH-Image program (available from http://www.ccp14.ac.uk/ccp/web-mirrors/nih-image/nih-image/download.html) as previously described.33
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Figure 1, a and b
, shows the
determination of an optimal primary antibody dose using anti-ICAM-1 as
an example. As described above, primary antibody binding was assessed
in the whole leukocyte population as well as to the neutrophil
subpopulation, the latter separately identified with Ly-6G staining.
The neutrophil subpopulation was assessed to monitor antibody binding
to low-expressed surface molecules or nonspecific binding at high
antibody doses. As can be seen from Figure 1a
, binding of anti-ICAM to
whole-blood leukocytes increased with primary antibody dose. Figure 1b
shows, however, that anti-ICAM-1 binding to neutrophils also occurred
at the higher dose (1.8 µg of ICAM-1/106
cells). Consequently, as labeling of neutrophils would lead to their
retention within the magnetic column, doses of primary antibodies that
showed an absence of, or very little binding to, neutrophils were
selected.
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Figure 1c
shows a cytospin of neutrophils isolated when
anti-ICAM-1 was absent from the primary antibody cocktail. The effect
of including anti-ICAM-1 in the antibody cocktail to remove
contaminating monocytes is clearly illustrated in Figure 1d
. The
isolation protocol routinely yielded 0.5 x
106
cells/ml whole blood representing a recovery
of 70% of circulating neutrophils. The final cell suspension was
typically >95% neutrophils.
We used flow cytometry to confirm the proportion of neutrophils before
and after negative immunomagnetic separation (Figure 2)
. Statistical analysis of the
neutrophil gate revealed that neutrophils represented 28% of
whole-blood murine leukocytes (Figure 2a)
. After negative
immunomagnetic separation, the same protocol and acquisition parameters
were used for the final cell suspensions and these were shown to be
>95% neutrophils. Residual contaminating cells in these suspensions
displayed characteristic forward and side-scatter properties of
monocytes (Figure 2b)
.
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Exclusion of trypan blue dye revealed that isolated neutrophils
were >97% viable. Flow cytometric analysis of isolated neutrophils
showed little change in surface L-selectin or CD18 expression compared
with neutrophils gated with anti-Ly-6G in whole blood (Figure 3, a and b
, respectively). However,
stimulation of isolated neutrophils with PMA
(10-7 mol/L) resulted in a reduction in surface
L-selectin and increase in CD18 expression (Figure 3, c and d)
demonstrating that although neutrophils were not activated by the
isolation procedure, they were still susceptible to activation by
applied stimuli. The PMA-stimulated increase of CD18 expression
reflects a similar up-regulation of CD11b on the cell surface (mean
fluorescence intensity: unstimulated whole-blood neutrophils =
109.49; PMA-stimulated whole blood neutrophils = 584.37;
unstimulated isolated neutrophils = 84.61; PMA-stimulated isolated
neutrophils = 737.17). These changes are characteristic of
neutrophil activation.35-38
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Exposure of isolated neutrophils to a chemoattractant in an
under-agarose assay caused a characteristic chemotactic migratory
response toward fMLP that was dependent on the dose of fMLP used
(Figure 4, A and B)
. Doses of 10 to 100
pmol (10 µl of 10-6 to
10-5 mol/L) induced significant
(P < 0.05) chemotaxis compared to control.
Optimal chemotaxis was induced by 30 pmol of fMLP, whereas higher doses
(300 pmol) reduced directed migration. These results are consistent
with the accepted hypothesis that high doses of chemoattractant can
desensitize leukocytes and result in an inhibition of neutrophil
orientation and chemotaxis.39,40
Other chemoattractants
have also been used in our laboratory to assess the ability of isolated
neutrophils to migrate under agarose. In our hands, studies
incorporating LTB4 have shown a maximal distance
of directed migration of 120 µm toward a source of 30 pmol (10
µl of 3 x 10-7 mol/L).
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Exteriorization of the cremaster muscle requires a small incision
in the scrotum through which the testis, epididymis, and the cremaster
that encloses them are gently extracted. A lateral incision from the
tip of the cremaster toward the point of exit from the scrotum allows
the cremaster muscle to be pinned across a glass coverslip while
superfused with a thermocontrolled bicarbonate buffer. This short
procedure rapidly induces leukocyte rolling that has been shown to be
predominantly P-selectin-dependent at 30 minutes.41
Histological examination of surgically stimulated vessels reveals that
>90% of the rolling population are neutrophils.42
Observation of isolated neutrophils in vivo was recorded at
30 minutes after surgical preparation. Fluorescent-isolated
neutrophils were observed to circulate for at least 30 minutes after
transfusion into recipient mice. Figure 5A
shows consecutive frames of two
isolated, fluorescently labeled neutrophils rolling in vivo.
QuickTime movies of these rolling cells are available on the website
(http://www.amjpathol.org). These images
illustrate the ability of isolated neutrophils to interact with the
postcapillary vascular endothelium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The negative immunomagnetic separation technique described above has
several distinct advantages over previously described neutrophil
isolation methods. The method of isolation we describe is based on
negative selection. The advantage of negative immunomagnetic separation
compared to a positive selection technique is that neutrophils are not
labeled with antibodies directed against their cell surface markers,
therefore reducing exposure to potential activating agents. In
addition, other workers have previously observed a high activation
status of human neutrophils isolated by density gradient centrifugation
compared to those isolated by immunomagnetic
separation.38,47
Density-gradient centrifugation, which is
the method of choice for isolation of neutrophils from humans and other
species, has inherent difficulties when it comes to isolation of murine
neutrophils. Variable levels of neutrophils and other leukocytes in the
peripheral circulation of mice,44,48
and insignificant
buoyancy differences between mouse neutrophils and lymphocytes hinder
the identification of distinct bands after density centrifugation.
Different cell types are therefore difficult to separate, leading to a
compromise between yield and purity (ie, retaining discrete parts of
the neutrophil band ensures purity but decreases potential yield). In a
recent study by Kruger and colleagues,49
isolation of
murine neutrophils from peripheral blood was abandoned as the numbers
of neutrophils yielded from density-gradient centrifugation was
insufficient. Kruger and colleagues,49
and also other
investigators,50
isolated neutrophils from bone marrow but
the maximal purity of the populations was lower than, or only as great
as, the minimal purity we achieve with immunomagnetic separation. Also,
the densities of gradients used for isolation of bone marrow
neutrophils from different strains of mice were not the same. Our
method overcomes these problems because total and differential cell
counts are made before isolation. Doses of each antibody are then
adjusted accordingly, maximizing the yield and purity of neutrophils
given by each preparation. We have specifically developed this
technique so that the amount of each antibody used is adjusted for the
number of cells it is directed against. This allows the method to be
used for any strain of mouse regardless of total leukocyte counts and
differentials. This is of practical importance when studying
genetically modified mice because deletion of certain molecules can
have profound effects on circulating neutrophils.51
Indeed, we have used the method to successfully isolate neutrophils
from C57BL/6 (>95% pure, yield 0.5 x
106
cells/ml whole blood), BALB/c (>97% pure,
0.5 x 106
cells/ml) and
interleukin-1ß-converting enzyme knockout
(ICE-/-) mice (>94% pure, 0.5 x
106
cells/ml).
To deplete monocytes from whole-blood leukocyte suspensions, F4/80 antigen was used as a monocyte-specific marker. As F4/80 antigen predominantly labels mature monocytes, anti-ICAM-1 was selected as a pan-lymphocyte/monocyte antibody to remove residual monocytes. ICAM-1 has been previously demonstrated to be expressed on circulating unstimulated rodent monocytes with negligible amounts expressed on neutrophils.30 We therefore postulated that careful administration of low doses of anti-ICAM-1 to the primary antibody cocktail would increase the purity of the neutrophil isolate without severely compromising yield. We found this to be a highly successful approach because addition of anti-ICAM-1 to the antibody cocktail increased the purity of the isolated neutrophil populations from 85 to >95%.
Although we have taken every possible measure to maximize yield, our
method gives 0.5 x 106
neutrophils per
ml of blood. Typically, 3 x 106
total
leukocytes per 1 ml whole blood was obtained. Based on blood smears
from the mouse strains used here, neutrophils represented 20 to 25%
of total leukocytes in the peripheral circulation. Therefore, our
method is able to retrieve 70 to 80% of neutrophils from whole
blood. Other workers have previously administered neutrophil-specific
inflammatory stimuli, such as thioglycollate17-19
to the
peritoneal cavity to obtain peritoneal lavage containing >95% pure
populations of neutrophils. This technique is elegantly simple, but is
limited by the fact that exposure of the neutrophils to an inflammatory
agent and migration into the peritoneum may alter the activity of
neutrophils in subsequent assays. It is now well established, for
example, that expression of surface molecules such as CD29
(ß1-integrin) and CD18
(ß2-integrin) are significantly increased after
neutrophil migration20-22
and that L-selectin is
shed.23,24
Our preliminary investigations indicate that neutrophils isolated using
negative immunomagnetic separation are viable for subsequent studies
in vitro and in vivo. We have used the
under-agarose migration assay to demonstrate the ability of isolated
murine neutrophils to respond to chemotactic stimuli including fMLP and
LTB4. Other studies that have used a variety of
chemoattractants to investigate the chemotactic ability of murine
leukocytes have shown fMLP to be a highly effective activating agent
for murine neutrophils.49
It was observed, however, that
mouse neutrophils migrated a smaller distance under agarose when
compared to human neutrophils under similar experimental conditions.
The migration of human neutrophils to fMLP under agarose in our hands
is consistent with published data.32,40
In contrast, the
average directed migration distance of murine neutrophils to an optimal
dose of fMLP was 10% of that of human neutrophils. Furthermore, the
optimal dose of fMLP that induced maximal migration of murine
neutrophils under agarose (30 pmol) is 30 times higher than that
previously observed, by ourselves and others, for human
neutrophils.40
Other workers have also reported data
supporting differential migratory behavior between human and murine
cells,19,40,52
suggesting that this phenomenon is because
of species difference. Nevertheless, isolated murine neutrophils are
clearly responsive and migrate under agarose toward a chemotactic
stimulus.
Our data obtained from in vivo experiments using isolated fluorescently labeled neutrophils show no significant difference in rolling behavior compared to endogenous cells. In addition, the rolling fraction of isolated neutrophils (33%) was similar to that of endogenous leukocytes routinely observed in vivo in our laboratory. Loading of isolated neutrophils with CFDA-SE is not believed to activate these cells as other workers have previously used this dye in studies of leukocyte activation53 and leukocyte rolling.34 Other workers have also compared the effects of various fluorescent dyes on neutrophil-endothelial cell interactions using rolling velocity as a sensitive parameter of neutrophil activation.54 That the rolling velocities of isolated neutrophils closely resemble endogenous neutrophils in vivo support the flow cytometry data suggesting that neutrophils have not been unduly activated by the isolation or the fluorescent labeling procedure. This conclusion is supported by additional flow cytometric observations in our laboratory of CFDA-SE-labeled cells that do not differ in forward-scatter characteristics compared to unstimulated neutrophils (data not shown). Shape change characteristics have been used in a number of studies investigating cell activation.55-57 Furthermore, fluorescent-labeled isolated neutrophils continued to circulate for at least 30 minutes after transfusion into the recipient mouse. This observation suggests that these cells are not activated by the isolation or labeling procedures.
Studying responses of genetically modified mice to inflammatory stimulation has already generated a wealth of information regarding precise molecular mechanisms of inflammatory responses.58-62 The ability to isolate neutrophils from one mouse and study their interactions in another mouse offers the potential to study genetic combinations. This can also be achieved by bone marrow transplantation although considerably more time is required. As mentioned, some strains of genetically modified mice, such as the CD18-/- and ICAM-1-/- mutants, have circulating neutrophil numbers significantly greater than those found in wild-type mice.44,48,51 Interpretation of data obtained using these mice with regard to neutrophil migration and inflammation is therefore made difficult. Isolation and labeling of neutrophils from these mutants, and injection into wild-type mice or vice versa would allow similar studies to be performed without such complications.
In summary, the isolation method described here provides the ability to study murine neutrophils within murine models, both in vitro and in vivo, thus eliminating the inherent questions of species differences, and providing more accurate models of disease. The data presented show negative immunomagnetic separation of murine blood can yield unactivated, viable, and responsive neutrophils.
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Footnotes |
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Supported by the British Heart Foundation (FS/99040), the Northern General Hospital Research Committee (RG004090), and the Wellcome Trust (057108, 042592, 043571).
Accepted for publication April 24, 2001.
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R. Boxio, C. Bossenmeyer-Pourie, N. Steinckwich, C. Dournon, and O. Nusse Mouse bone marrow contains large numbers of functionally competent neutrophils J. Leukoc. Biol., April 1, 2004; 75(4): 604 - 611. [Abstract] [Full Text] [PDF]
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