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(Journal of Leukocyte Biology. 2001;69:497-504.)
© 2001 by Society for Leukocyte Biology

Leukocyte-specific gene 1 protein (LSP1) is involved in chemokine KC-activated cytoskeletal reorganization in murine neutrophils in vitro

Michael Hannigan, Lijun Zhan, Youxi Ai and Chi-Kuang Huang

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte-specific gene 1 protein (LSP1) is a cytoskeletal-associated protein of leukocytes that in vitro cross-links F-actin into extensively branched bundles of mixed polarity. In this study, we examined chemotaxis and superoxide production in neutrophils prepared from wild-type (WT) and Lsp1 knockout mice. Compared to WT neutrophils, Lsp1-/- neutrophils showed impairment in both migration speed and chemotaxis direction during chemokine KC-directed chemotaxis. When examined by confocal microscopy, chemotaxing Lsp1-/- neutrophils showed abnormal morphologies. They had discontinuous primary actin-rich cortexes and large membrane protrusions. When stimulated by phorbol 12-myristate 13-acetate (PMA), Lsp1-/- peritoneal neutrophils produce more superoxide than WT. The data presented suggest that LSP1 plays important roles in the regulation of neutrophil morphology, motility, and superoxide production.

Key Words: neutrophil • activation • knockout mice


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The morphology of crawling cells in chemotactic gradients has been described for neutrophils, fibroblasts, and other migratory cells. On a two-dimensional substrate, cell morphology is determined by the organization of the underlying actin cytoskeleton [1 2 3 ]. The cytoskeleton in turn is shaped by the interaction of actin filaments and filament cross-linking proteins [4 5 6 ]. Membrane protrusions are used for sensing and movement [7 ], whereas the actin-rich cytoskeleton surrounds the periphery of the cell and provides rigidity [8 ].

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Lsp1-/- mice [28 ] were transferred from the University of Toronto to the University of Connecticut Health Center and maintained in the Center for Laboratory Animal Care. Wild-type (WT) 129/SvJ mice were purchased from Jackson Laboratories and bred in the Center for Laboratory Animal Care to obtain age-matched controls. The University of Connecticut Health Center animal care committee approved all procedures involving mice.

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.



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  		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 ({blacktriangleright}), 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, {blacktriangleright}), and a constriction ring (frame 15, {triangleright}). Shown in B are the structures observed on Lsp1-/- neutrophils. They include a tubule-like structure (frame 1, {star}), lamellipodium (frame 6, ) multiple anterior membrane protrusions (frame 11, ), uropod (frame 13, {blacktriangleright}), and a non-contiguous constriction ring (frame 16, {triangleright}).

 


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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 ({blacktriangleright}), 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.

 
Confocal microscopy of migrating neutrophils
Slides containing chemotaxing neutrophils were prepared by incubation in Zigmond chambers containing KC gradients for 15 min at 37°C. The buffer solutions in both sides of the chamber were then removed and immediately replaced with 100 µL of 2% paraformaldehyde in phosphate-buffered saline (PBS) [33 ]. After 10 min of fixation, coverslips were removed. Fixed neutrophils on coverslips were permeabilized with 0.01% lysolecithin in PBS (PBS-lysolecithin) and incubated with FITC-phalloidin (1:50 dilution in PBS-lysolecithin for 20 min at room temperature). Finally, the coverslips were washed with PBS-lysolecithin and mounted with Slow Fade reagent. Slides were examined using a Zeiss confocal imaging system at the Center for Biomedical Imaging Technology, University of Connecticut Health Center. The samples were excited with 488-nm-wavelength light from an argon/krypton laser. Detection was done with filters for 515-nm light.

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Colocalization of LSP1 and F-actin in WT neutrophils undergoing chemotaxis
WT neutrophils undergoing chemotaxis toward chemokine KC were processed for immunofluorescence of LSP1 and F-actin and examined by confocal microscopy. A representative neutrophil is shown in Figure 1A . In the left and central panels, LSP1 is shown in green and F-actin in red, respectively. LSP1 was found to co-localize with F-actin in the filopodia, lamellipodia, ruffles, and the actin-rich cell cortex. In contrast, LSP1 was not found in the retraction fibers that contained F-actin. As controls, samples were also stained for only LSP1 or F-actin and analyzed in the same manner. Representative examples are shown in Figure 1B . No rhodamine fluorescence was observed when cells were stained with fluorescein, nor was fluorescein fluorescence observed in samples stained with rhodamine.

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).



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  		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).

 
LSP1 was detected in the WT but not Lsp1-/- neutrophils as shown by immunostaining (Fig. 2A) and immunoblot (as a 52-kDa protein [14 ]; Fig. 2B ). F-actin polymerization induced by KC was similar both in WT and Lsp1-/- neutrophils (Fig. 2C) . However, superoxide production induced by PMA was higher in the Lsp1-/- (36.7 ± 12 nmol/107 cells) than WT (13.8 ± 4 nmol/107 cells) neutrophils (Fig. 2D) . Immunoblotting with antibodies to p47phox, p67phox, gp91phox, and p22phox showed no significant difference in the amounts of oxidase proteins expressed in the WT and Lsp1-/- neutrophils (data not shown).

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) .



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  		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.

 
Morphology of migrating Lsp1-/- neutrophils
The processes of anterior lamellipodial extension, cytoplasmic streaming, and posterior retraction are observed as specific morphological structures of motile cells [41 ]. Lamellipodium extended to various lengths on chemotaxing WT neutrophils (Fig. 4A , indicated in frame 4). Cytoplasm streamed into portions of lamellipodia and formed broad tubules (Fig. 4A , indicated in frame 6). In the neutrophil pictured (Fig. 4A) , a uropod was transiently observed (Fig. 4A , frames 9–11). Bright constriction rings caused by cortical contraction were also seen (Fig. 4A , frame 15) on the cell surface.

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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  		Table 1. Increased Cell Protrusions and Decreased Lamellipodia Area on KC-Stimulated Lsp1-/- Neutrophils

 
To demonstrate that this was not simply an isolated cell, at least 100 WT and Lsp1-/- neutrophils were examined by confocal microscopy and their shapes classified based on the system of Keller [42 ]. Of the stimulated WT neutrophils, 85.8% were front-tail polarized and the remaining 14.2% were unifocal (spherical with one projection). In contrast, when stimulated Lsp1-/- neutrophils were examined 20.7% were spherical rough, 3.4% were unifocal, 21.8% were non-polar with projections, 54% were elongated with multiple protrusions, a classification not described by Keller, and none were front-tail polarized.

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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we demonstrate that compared with WT neutrophils, Lsp1-/- neutrophils showed abnormal morphology and impairments in migration speed and direction in KC gradients, but increased superoxide anion production by PMA stimulation. No significant differences in adhesion, degranulation, and F-actin polymerization induced by KC were observed. The production of superoxide anion and F-actin polymerization after KC stimulation indicates that the signal pathway through the KC receptor is functional. Chemotaxis and superoxide production are also critically dependent on cytoskeletal organization [2 , 40 , 43 ]. An altered cell cortex could at least partially account for the phenotypes observed in Lsp1-/- neutrophils. Cross-linking of F-actin regulates the viscosity and stiffness of the actin-rich cell cortex [4 , 13 , 44 ]. Thus, a loss of LSP1 in neutrophils may produce a less stable actin-rich cell cortex with lower viscosity and less stiffness. It is likely that Lsp1-/- neutrophils are unable to properly bundle actin during chemotaxis and this affects neutrophil motility and morphology.

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 {alpha}-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.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant AI-20943. LSP1-/- mice were a generous gift from Dr. Jan Jongstra and Victoria Misener of Toronto Western Research Institute, Toronto Ontario Canada. The authors would like to thank the staff of the Center for Biomedical Imaging Technology at the University of Connecticut Health Center for their guidance during the preparation of this article.

Received September 9, 2000; revised October 18, 2000; accepted October 20, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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