Department of Pathology, University of Connecticut Health Center, Farmington
Correspondence: Michael Hannigan, Department of Pathology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3105. E-mail: michaelhanniga{at}snet.net
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neutrophil • activation • knockout mice
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of the various internal and external forces that shape the actin cytoskeleton are mediated by F-actin binding proteins [9 10 11 ]. These proteins directly influence cell morphology and motility because of the cross-linking angles and rigidity of filament bundles they form [12 , 13 ].
Leukocyte-specific gene 1 protein (LSP1) is an F-actin bundling protein found in macrophages, B lymphocytes, T lymphocytes, and neutrophils [14 15 16 17 18 19 ]. The amino-terminal half of murine LSP1 contains two putative Ca2+ binding domains and is rich in acidic amino acids [14 ]. The carboxy-terminal half is rich in basic amino acids and contains at least one high-affinity F-actin binding site [20 ]. Mouse and human LSP1 are highly homologous [21 , 22 ], suggesting an evolutionary conserved function. In vitro, LSP1 forms F-actin into thick bundles of mixed polarity with multiple branches [23 ]. It is a substrate of protein kinase C in T lymphocytes [24 ] and B lymphocytes [25 ] and a major substrate of MAP kinase-activated protein kinase 2 (MAPKAP kinase 2 or MK2) in neutrophils [26 ]. It associates with membrane IgM [27 ] and regulates anti-IgM-induced apoptosis of immature B cells [28 ]. In neutrophils of patients with NAD 47/89 LSP1 is overexpressed [29 ] and these neutrophils demonstrate deficiencies in migration, phagocytosis, and spreading [30 ].
LSP1 knockout mice have recently been prepared [28 ]. In this study, we examined the chemotaxis and superoxide production of Lsp1-/- neutrophils prepared from these mice.
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The following materials were purchased: crystallized bovine serum albumin (BSA), lysolecithin, murine fibrinogen, and normal goat serum from Sigma Chemical (St. Louis MO); rhodamine and fluorescein isothiocyanate (FITC) conjugates of phalloidin and Slow Fade reagent from Molecular Probes (Eugene, OR); peroxidase and FITC conjugates of goat anti-rabbit immunoglobulin from Jackson Immunoresearch (West Grove, PA); murine KC from R & D Systems (Minneapolis, MN); NIM2 reagents from Cardinal Associates (Santa Fe, NM); enhanced chemiluminescence (ECL) reagent from Amersham (Piscataway, NJ); and biotinylated anti-CR1 antibody and streptavidin-FITC from PharMingen (San Diego, CA).
Preparation of neutrophils and chemotaxis chamber assays
Peripheral blood neutrophils were purified using NIM 2
reagents from anticoagulated blood (0.05 M EDTA) obtained by cardiac
exsanguination. Neutrophils were suspended in Hanks’ buffer (0.14 M
NaCl, 5.4 mM KCl, 1 mM Tris, 1.1 mM CaCl2, 0.4 mM
MgSO4, 1 mM HEPES, pH 7.2) containing 5 mg/mL BSA. After
isolation, cells were allowed to adhere to glass coverslips for 5 min
at 37°C. The coverslips were then rinsed and placed on Zigmond
chambers (Neuroprobes, Cabin John, MD) [31
]. Aliquots
(0.1 mL) of a solution (Hanks’ buffer containing a 1:10 dilution of
10% gelatin in H2O) were added to one side of the chamber
and the same solution containing mouse KC (1 µg/mL) was added to the
other side. Chambers were then used for videomicroscopy or
immunohistochemistry.
Determination of neutrophil directionality and speed
Time-lapse videomicroscopy was used to examine neutrophil
movements in Zigmond chambers [32
, 33
]. The
microscope was equipped with differential interference contrast optics
and a x10 objective. Images were captured at 1-s intervals with a
PXL-EEV37 CCD camera and ISEE analytical imaging software.
Videomicroscopy of migrating neutrophils
The microscopy equipment described above with a x63 objective
was used to examine individual cell morphologies during migration. NIH
image (http://rsb.info.nih.gov/nih-image) and Adobe PhotoShop software
were used to subtract backgrounds and photographically enlarge original
images. For reference, a 10-µm bar is shown in Figure 1
, and a 20-µm bar in Figures 4
and 5
. Measurements of lamellipodia
area were obtained from original images through the use of the NIH
image software.
 View larger version (84K): [in a new window] |
Figure 1. (A) Serial optical sections from confocal images localizing LSP1 and
F-actin in a chemotaxing neutrophil. This peripheral blood neutrophil
is undergoing chemotaxis to the left in response to KC (1 µg/mL at
high end). The left panels are optical sections 1 µm apart beginning
at the coverslip showing LSP1 localization in green. Middle panel, the
same cell and focal planes showing F-actin in red. The right panel is
the merged view showing colocalization in yellow. The following
morphological features are indicated in the merged view: lamellipodia
( ), filopodia ( ), actin-rich cell cortex ( ), and retraction
fibers (). A 10-µm bar is shown in the first frame of each panel.
(B) Control images of WT neutrophils stained for either LSP1 or
F-actin. Neutrophils undergoing chemotaxis were stained for LSP1 (top
panels) or F-actin (bottom panels). Images obtained through the
fluorescein channel are shown on the right, whereas those from the
rhodamine channel are on the left.
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Figure 4. Individual frames from time-lapsed video images of
Lsp1-/- neutrophils during chemotaxis in Zigmond
chambers. Time-lapse videomicroscopy was used to examine WT (A) or
Lsp1-/- (B) neutrophils in the process of
chemotaxis toward KC (1 µg/mL). Each frame was obtained 4 s
after the previous frame. In both A and B the neutrophil migrated
across the frame from left to right. The bar represents 20 µm. In A,
typical morphological structures observed on WT neutrophils are
indicated. These include lamellipodium (frame 4, ), filopodia (frame
6, ), uropod (frame 9, ), and a constriction ring (frame 15,
 ). Shown in B are the structures observed on
Lsp1-/- neutrophils. They include a tubule-like
structure (frame 1,  ), lamellipodium (frame 6, ) multiple
anterior membrane protrusions (frame 11,  ), uropod (frame 13, ),
and a non-contiguous constriction ring (frame 16, ).
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Figure 5. Confocal images of F-actin in WT (A) and Lsp1-/-
(B–D) neutrophils. Neutrophils seeded on coverslips were incubated for
15 min in a chemotactic gradient (1 µg/mL KC) in Zigmond chambers.
Fixed neutrophils were stained with FITC-phalloidin to visualize
F-actin with a confocal microscope. Frame 1 of each set is the focal
plane at the level of the coverslip. Each subsequent frame is separated
by a 1-µm increment. In each panel F-actin-containing cell structures
are marked as follows: lamellipodia (), actin-rich cell cortex
(), retraction fibers (), filopodia (), uropod (), and
ruffles (). In addition Lsp1-/- neutrophils
have well-separated multiple protrusions (B panel 1, and D panels 1 and
2,  ). A 20-µm bar is included in the first frame of each
panel.
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To analyze both LSP1 and F-actin, coverslips containing neutrophils from Zigmond chamber assays were incubated with a solution of PBS-lysolecithin containing normal goat serum (1%) and rabbit anti-mouse LSP1 serum (1:100 dilution) [26 ] for 30 min. They were then incubated in PBS-lysolecithin containing FITC-conjugated goat anti-rabbit immunoglobulin (1:100 dilution) and rhodamine-phalloidin (1:50 dilution). Coverslips were next mounted with a drop of Slow Fade reagent and examined using the Zeiss confocal system. Excitation wavelengths of 488 nm and 568 nm were used with emission filters to detect FITC (515–520 nm) and rhodamine (590 nm).
Superoxide production
Peritoneal neutrophils were obtained 16 h after an
intraperitoneal injection of 2.4% thioglycollate (1 mL). These
neutrophils (1–2 x 105) were placed into the wells
of a microtiter plate containing a reaction mix composed of 145 µM
cytochrome c, 2 mM sodium azide, 2 mM CaCl2, and
2.4 mM MgCl2. Phorbol 12-myristate 13-acetate (PMA; 0.1
µg/mL) or buffer was added to the appropriate wells and the plates
incubated at 37°C for 30 min. During the incubation, the reduction of
cytochrome c was measured at 550 nm with a Molecular Devices
thermomax plate reader (Menlo Park, CA) and Softmax analysis software
(Molecular Devices) as described [34
]. The maximum
superoxide anion produced was also calculated [35
].
Immunoblots
The peritonea of WT and Lsp1-/- mice were
rinsed to obtain neutrophils 4 h after intraperitoneal injection
of thioglycollate (1 mL, 2.4%). Neutrophils (2 x
105) were lysed with sample buffer, boiled, and resolved in
10% polyacrylamide gel electrophoresis (PAGE) [36
].
Samples were then analyzed as immunoblots using rabbit anti-murine LSP1
serum (1:1000 dilution) followed by peroxidase-conjugated goat
anti-rabbit immunoglobulin. Bound antibodies were visualized using the
ECL detection system.
Flow cytometry
Peripheral blood neutrophils (1 x 106)
suspended in HBSS were stimulated with KC (1 µg/mL). Samples were
removed at the indicated times and immediately fixed by the addition of
2% paraformaldehyde. To measure F-actin content, fixed samples were
permeabilized and stained for F-actin with FITC phalloidin (1:50) in
PBS-lysolecithin [37]. To measure CR1 levels as a marker of
degranulation [38
], samples were incubated for 20 min
with biotinylated anti-CR1 antibodies (1:50) for 20 min at room
temperature. After washing, the samples were then stained for 20 min
with FITC-streptavidin (1:100). All samples were analyzed on a
Becton-Dickinson FACScan instrument using the FL-1 channel and Cell
Quest software.
Adhesion
Neutrophil adhesion to fibrinogen-coated microtiter plates was
performed as described previously [39
]. One hundred
microliters of bone marrow cells suspended in Hanks’ buffer (3 x
106 cells/mL) were plated onto microtiter plates previously
coated with fibrinogen (100 µg/mL, 16 h at 4°C). After a
60-min incubation, 100 µL of medium was removed from the top of the
wells and the plates were centrifuged (5 min, 60 g)
upside-down on 3M paper in a swing bucket rotor. The percentage of
adherent cells was determined by measuring membrane acid phosphatase
[40
]. Enzyme activity was assayed in parallel on 100
µL of the original cell suspension to determine the 100% reference
value.
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Characterization of WT and Lsp1-/-
neutrophils
Both the WT and Lsp1-/- resting neutrophils
showed round morphology with similar cell size and F-actin content
(Fig. 2A
). The equivalence of F-actin content in resting cells was also
confirmed by FCM. No difference in the average mean fluorescence of
nontreated WT (165.6 ± 63) and Lsp1-/-
(184.4 ± 89) was observed (n = 8).
 View larger version (26K): [in a new window] |
Figure 2. Characterization of WT and Lsp1-/-
neutrophils. (A) Confocal images of resting peripheral blood
neutrophils from WT and Lsp1-/- mice. In the top
panels (frames 1 and 2) a focal plane from a WT neutrophil is shown,
whereas the bottom panels are from a Lsp1-/-
neutrophil. Frame 1 shows LSP1 staining in green, frame 2 is the red
F-actin staining. In the bottom panel (frames 3 and 4) no LSP1 staining
is observed in frame 3, indicating that the cell does not express LSP1.
F-actin is observed as red staining in frame 4. (B) Immunoblot
detection of LSP1 in peritoneal neutrophils. Peritoneal neutrophils
(2 x 105) were analyzed by immunoblotting. Those
samples obtained from Lsp1-/- mice (lane 1) did
not contain anti-LSP1 reactive proteins, whereas those from WT mice
(lane 2) did. (C) Actin polymerization in WT and
Lsp1-/- neutrophils stimulated with KC.
Neutrophils in suspension were stimulated with KC (1 µg/mL) and
levels of F-actin were measured kinetically by FCM as described in
Materials and Methods (n = 4). (D) Superoxide
production by WT and Lsp1-/- neutrophils induced
by PMA, chemokine KC, or buffer. Peritoneal neutrophils (1–2 x
105) were stimulated with PMA (0.1 mg/mL) or KC (5 µg/mL)
and the production of superoxide was measured for 30 min.
**Significantly more superoxide produced compared to WT stimulated with
PMA at the P < 0.02 level (n = 4). (E)
Adherence of WT and Lsp1-/- cells in response to
buffer treatment, PMA, or KC stimulation. Adherence to
fibrinogen-coated plates was measured, and the percent adherent at 10,
30, and 60 min plotted (n = 3). (E) CR1 expression
induced by KC. The mean fluorescence of neutrophils stained with
anti-CR1 antibodies was determined 2.5, 5, and 10 min with or without
KC stimulation (n = 2).
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The percent of adherent of Lsp1-/- neutrophils compared to WT neutrophils, and the kinetics of their adherence was not different after PMA or KC stimulation (Fig. 2E) . In addition, no difference in degranulation was noted by KC stimulation as measured by CR1 up-regulation (Fig. 2F) .
Motility analysis of WT and Lsp1-/-
neutrophils in vitro
Plots of the final position of neutrophils relative to a
common origin (Fig. 3
) were constructed from the data obtained from video images of WT
and Lsp1-/- neutrophils undergoing chemotaxis in
Zigmond chambers. This analysis showed that the speed of
Lsp1-/- neutrophils (18.5 ± 0.5 µm/min)
was reduced compared to WT (30.8 ± 0.9 µm/min). Movement up the
KC gradient was also impaired compared to WT neutrophils (Fig. 3)
.
 View larger version (15K): [in a new window] |
Figure 3. Plots of WT (A) and Lsp1-/- (B) neutrophil
migration in the Zigmond chamber assay. Neutrophils (106
cells/mL) undergoing chemotaxis in a Zigmond chamber in response to KC
(1 µg/mL) were recorded using time-lapse photography. Tracings of
individual neutrophils were used to plot a final position relative to a
common starting position. Positive X values represent movement up the
gradient, whereas absolute Y values represent lateral movement.
Circular histograms indicating the percentage of cells distributed in
each 10° angle are shown as insets.
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These typical morphological features were not observed as often in chemotaxing Lsp1-/- neutrophils (Fig. 4B) . The large glass-like lamellipodia observed on WT neutrophils were rarely seen on Lsp1-/- neutrophils. Lsp1-/- neutrophil lamellipodia, when present, were small (37% less area, Table 1 ) and transient compared to WT lamellipodia. Instead of typical lamellipodia, the primary pseudopodia of chemotaxing Lsp1-/- neutrophils were tubule-like structures. Forward movement appeared to occur in Lsp1-/- neutrophils by extension of several of the tubules at once (Fig. 4B , frame 11). This gave the unusual feature of a neutrophil with multiple distinct anterior protrusions. Lsp1-/- neutrophils were observed to have almost five times more filopodia/tubule-like protrusions per cell compared to WT (Table 1) . Lateral movement of the Lsp1-/- neutrophil uropods, as observed in Figure 4B (frames 13–16), suggests that unlike WT, the Lsp1-/- uropods are not in contact with the coverslip. Finally, the constriction rings on Lsp1-/- neutrophils were not contiguous as they were on WT neutrophils (Fig. 4B frame 16).
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View this table: [in a new window] |
Table 1. Increased Cell Protrusions and Decreased Lamellipodia Area on
KC-Stimulated Lsp1-/- Neutrophils
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Confocal images of F-actin structures of WT and
Lsp1-/- neutrophils
F-actin in WT (Fig. 5A
) and Lsp1-/- (Fig. 5B
5C
5D)
neutrophils
was examined by confocal microscopy using FITC-phalloidin. Optical
sections of individual cells allowed the examination of F-actin at
various levels of the cell relative to the substratum. In the
representative neutrophils shown, frame 1 in each panel represents the
most ventral image of the cell showing contact with the coverslip. In
WT neutrophils the expected F-actin structures were observed. Some of
the common F-actin structures of migrating WT neutrophils are pointed
out in Figure 5A
.
Many of the Lsp1-/- neutrophils had multiple cellular protrusions with features of leading fronts. In the first representative neutrophil (Fig. 5B , frames 1 and 2), one of the two (left) anterior protrusions seems to be dominant in that it contains filopodia and small membrane ruffles. This protrusion is also thicker because it is not lost when the focus is shifted dorsally. There is a third protrusion located near the posterior end of the cell but at a right angle to the two previous protrusions (Fig. 5B , frames 1 and 2). It contains small filopodia and ruffles. Because it has three well-separated membrane protrusions, it is difficult to discern which way this cell is moving.
Like many of the Lsp1-/- neutrophils observed, the neutrophil in Figure 5C shows numerous punctuate bright-staining actin structures visible in frame 1. As the focus is shifted dorsally, this neutrophil also appears to have two fronts (Fig. 5C frames 3 and 4), each containing membrane ruffles and filopodia. In this case the apparent fronts are in direct line with each other and separated by a 3.5-µm distance.
The neutrophil shown in Figure 5D exemplifies another common feature of LSP1-/- neutrophils. This neutrophil has a rounded cell body with little elongation. In the first frame, two anterior protrusions are seen, as are numerous punctuate bright-staining actin structures. As the focus is shifted dorsally, one of the protrusions disappears very rapidly, indicating that it is not very thick. Another unusual feature observed in this cell and other LSP1-/- neutrophils is the uropod, which is not attached to the coverslip. This is apparent because it is located in frames taken at higher focal planes where the cell front is absent.
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The colocalization of LSP1 with F-actin in WT neutrophils and the
morphological differences between WT and Lsp1-/-
neutrophils suggests that it may regulate the stability and turnover of
F-actin during chemotaxis. Within cells there are two types of F-actin
that differ in their stabilities. The lamellipodia F-actin is sensitive
to chemotactic factor stimulation and cytochalasin B treatment. In
contrast, cortical F-actin is more stable, present in both stimulated
and nonstimulated neutrophils and is cytochalasin B-insensitive
[8
]. The difference in stability of F-actin in lysed
neutrophils is likely controlled by actin filament binding proteins
such as 
-actinin, tropomyosin, and ABP [45
]. These
proteins may also regulate the rapid turnover of F-actin in the
lamellipodia [46
].
Superoxide production involves the assembly of several proteins to form an active NADPH oxidase on the plasma membrane [47 ]. During the assembly of the oxidase, cytosolic proteins p47phox and p67phox must translocate from the cytoplasm to the plasma membrane. In the case of Lsp1-/- neutrophils, the submembrane barrier to this translocation may be reduced, as observed by the apparent discontinuity of the cell cortex. Thus, a higher degree of active oxidase assembly may be achieved upon stimulation with PMA, which results in a higher level of superoxide production.
Recently we identified a single phosphorylation site for MK2 and multiple phosphorylation sites for protein kinase C in LSP1 (data not shown). Both MK2 and protein kinase C are known to be activated by chemotactic factors [48 ]. Further work is required to study the possible roles of LSP1 and phospho-LSP1 in neutrophil activation, particularly their interaction with the two types of F-actin in neutrophils. The results presented in this study suggest that LSP1 plays important roles in the regulation of neutrophil morphology, motility, and superoxide production.
Received September 9, 2000; revised October 18, 2000; accepted October 20, 2000.
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