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IMMUNOBIOLOGY
From the Toronto Western Research Institute, Cell and
Molecular Biology Division and Department of Immunology, Division of
Respirology, Department of Medicine, University of Toronto, and Toronto
General Division of the University Health Network, Toronto,
Canada; New York University Medical Center, Skirball Institute of
Biomolecular Medicine, Developmental Genetics Program, New York, NY.
Lymphocyte-specific protein 1, recently renamed leukocyte-specific
protein 1 (LSP1), is an F-actin binding protein expressed in
lymphocytes, macrophages, and neutrophils in mice and humans. This
study examines LSP1-deficient (Lsp1 Mouse lymphocyte-specific protein 1 (LSP1;
p50, pp52) is an intracellular phosphoprotein of 330 amino acid
residues expressed in B-lineage cells, functional T cells and
thymocytes, and macrophages and neutrophils derived from fetal liver
and bone marrow.1-4 LSP1 is not expressed in other
hematopoietic lineages such as erythrocytes, mast cells, or
megakaryocytes. In addition, nonhematopoietic tissues such as liver,
kidney, heart, lung, brain, skeletal muscle, and testis do not express
LSP1 messenger RNA (mRNA).1,3 Human LSP1 (WP34) has a
similar pattern of expression and shares a significant sequence
homology with mouse LSP1 particularly in the carboxyl-terminal half of the molecule.5-7 LSP1 contains a high-affinity
F-actin binding site, which directs it to the microfilament-rich
cortical cytoskeleton directly underneath the plasma
membrane.2,8,9 LSP1 is a substrate for protein kinase C
(PKC) and for MAPKAP kinase 2.10-12 LSP1 has 2 putative
EF-hand Ca++-binding motifs near the amino terminus and
recombinant LSP1 binds Ca++.2
Evidence suggests that LSP1 is an important signaling molecule
that regulates cytoskeletal architecture and motility as well as
receptor-induced apoptosis. Howard and coworkers13 showed that LSP1 expression in various eukaryotic cell lines, including the
highly motile melanoma cell line A7, resulted in the development of
hairlike projections that contain thick bundles of F-actin filaments.
LSP1-expressing A7 cells also exhibited a lack of motility even when
the level of LSP1 expression was not sufficient to produce hairlike
formations. These findings support the earlier suggestion that LSP1
overexpression may be responsible for the morphologic and motile
abnormalities of neutrophils of patients with the neutrophil actin dysfunction disorder, NAD 47/89, which exhibit a lack of motility
and numerous F-actin-rich hairlike projections on their surface.14,15 We recently found that LSP1 plays a role in
anti-IgM-induced apoptosis of the B-cell lymphoma line WEHI-231 and
immature splenic B cells.16 It is unknown whether the 2 functions of LSP1, that is, regulation of the cytoskeleton-driven
events and receptor-induced apoptosis, are mechanistically related or
work independently in a cell type-specific manner.
Based on the biochemical and functional characteristics of LSP1,
its cytoskeletal localization in the proximity of the plasma membrane,
and its restricted hematopoietic expression, we postulated that LSP1 is
important for the proper functioning of the immune system. To
investigate the role of LSP1 in vivo we generated an LSP1-deficient
(Lsp1 Gene targeting and production of Lsp1
Antibodies for flow cytometry
Cell collection and flow cytometric analysis
Adhesion of peritoneal macrophages to fibronectin Peritoneal cells were washed and resuspended (4 × 106 cells/mL) in RPMI media supplemented with 10% FCS, 2 mmol/L glutamine, penicillin/streptomycin (100 U/mL), and 0.1 mmol/L nonessential amino acids (all from Life Technologies, Gaithersburg, MD). Cells (1-4 × 105) were cultured in 24-well plates containing fibronectin-coated coverslips for 2 hours at 5% CO2, washed twice with RPMI media, and fixed in methanol for 10 minutes at room temperature. Immunostaining with biotinylated antimouse F4/80 antibodies (1:75, Serotec) and streptavidin-peroxidase was performed using Universal Immunostaining kit (Immunotech, Marseille, France) as described by the supplier. No staining was observed with isotype control. F4/80+ cells were enumerated in a blinded fashion within 10 randomly chosen area units (defined by the area of a grid, 0.0625 mm2). Cell counts from wells containing similar numbers of macrophages (as determined by flow cytometry, see above) were compared between wt and Lsp1 /strains.
Bronchoalveolar lavage and cell counts Mice were anesthetized by a single intraperitoneal injection of pentobarbital (Somnotol). Alveolar cells from lungs were lavaged 3 times with 0.6 mL PBS using an angiocath (22 gauge) inserted into the trachea. Cells were cytocentrifuged, fixed with methanol, and stained with May-Grunwald-Giemsa. The percentage of macrophages was determined by counting a total of 400 cells/slide. Total cell numbers were counted after resuspending the cells in HF (1-2 × 106 cells/mL).Neutrophil chemotaxis assay Peritoneal exudates from wt and Lsp1/mice, containing 80% neutrophils, were
obtained 4 hours after TG injection by lavage with Ca++-,
Mg++-free HBSS. Cells were labeled with 1.5 µmol/L
calcein-AM for 30 minutes at room temperature in HBSS and
washed twice with Ca++-, Mg++-containing HBSS
with 0.1% bovine serum albumin (BSA). Microplate chambers (96 wells)
with 3 µm pore polycarbonate filters from Neuro Probe, Inc (Cabin
John, MD) were used for chemotaxis. Chemoattractants (106-104 mol/L N-formyl
methionyl-leucyl-phenylalanine [fMLP], Sigma, or 106
mol/L C-X-C chemokine [KC], R&D Systems, Minneapolis, MN) were placed
in the lower chamber and 25 µL of cells (1 × 105) were
placed on top of the chamber and incubated for 45 minutes at 37°C in
a tissue culture incubator with 5% CO2. After incubation, nonmigrating cells were aspirated gently from the upper side of the
filter, which was further wiped clean using cotton swabs. Fluorescence
of the underside of the filter was determined using a Cytofluor 2300 (Millipore) fluorescent plate reader. The number of neutrophils that
migrated across the filter was quantified using a standard curve (cell
number versus fluorescence) constructed separately for neutrophils from
each mouse. All assays were done in triplicate.
Immunization and measurement of ovalbumin (OVA)-specific antibody production Mice were immunized by a single intraperitoneal injection of 50 µg OVA (Sigma) in complete Freund's adjuvant (Life Technologies). Mice were bled from the tail vein at various time points following immunization and OVA-specific antibody titers were measured by enzyme-linked immunosorbent assay (ELISA).28 Plates were developed with p-nitrophenyl phosphate substrate (Sigma FAST pNPP, Sigma) and optical densities at 405 nm were read on a plate reader (EL311sx AutoReader, Bio-Tek Instruments, Winooski, VT). The same anti-OVA serum sample was used to generate a linear standard curve in all experiments. Anti-OVA titers are expressed in arbitrary units relative to the standard.Statistical analysis All data are expressed as mean values ± 1 SEM. Unpaired Student t test or Welch t test was used with GraphPad InStat software (San Diego, CA).
Confirmation of Lsp1 (F1) mice produced
Lsp1/ mice at the expected mendelian
frequency in the F2 generation. To confirm the absence of LSP1
expression in Lsp1/ mice, cell lysates
prepared from bone marrow, thymus, spleen, and peritoneal cavity were
analyzed by Western blotting. As shown in Figure 1C, the typical LSP1
doublet bands2,29 are detected in lysates from wt mice, but
not from Lsp1/ mice. Actin levels indicate
equivalent loading. The LSP1 gene encodes 2 isoforms, LSP1
and S37, which share the amino acids 24 to 330.17 The
anti-LSP1 polyclonal antiserum used recognizes the C-terminal half of
LSP1, amino acids 179 to 330,16 which is identical in the
2 isoforms. Figure 1C confirms that the hematopoietic tissues tested
from Lsp1/ mice do not express S37 as
expected,17 hence, S37 cannot compensate for LSP1
deficiency in hematopoietic cells. Lsp1/
mice thrive and reproduce as well as their wt counterparts and mice
observed for up to 14 months are grossly normal with no obvious health
defects. No differences are observed in body weight and in cellularity
of spleen, thymus, lymph nodes, peritoneal cavity (see below), or
bone marrow.
An expanded population of peritoneal macrophages in
Lsp1 /
and wt mice in various tissues by flow cytometry. As shown in Figure
2, the proportion of myeloid cells in
bone marrow and neutrophil and monocyte populations in blood and spleen
are similar in the 2 genotypes, indicating that LSP1 deficiency does
not affect myeloid development. In contrast, peritoneal
Mac-1hiCD5 macrophages are expanded
significantly in Lsp1/animals. Figure
3 illustrates a typical analysis where
staining for Mac-1 and CD5 showed a 3-fold increase in the proportion
of Mac-1hiCD5 cells, whereas the proportion
of Mac-1hiCD5+ cells was similar to wt, as seen
in panels A and B. To confirm that Mac-1hi cells analyzed
are macrophages, we stained the peritoneal cells for Mac-1 and the
macrophage marker F4/80, which is expressed on the surface of both
CD5 and CD5+ subsets of
macrophages.30-32 Panels C and D show that all
Mac-1hi cells are F4/80+, and thus are
macrophages. The proportion of total F4/80+ macrophages is
increased by approximately 2-fold in the peritoneum of
Lsp1/ mice. The expansion of macrophages is
also detected by FSC/SSC analysis. Panels E and F illustrate the
presence of an expanded population of peritoneal cells (M), which are
larger and more granular than the lymphocytes (L). When
Mac-1hiCD5or
Mac-1hiF4/80+ cells (shown in panels A-D) were
gated and analyzed for their FSC/SSC properties, the cell population
corresponding to M in the total peritoneal cells was found (compare
panels G and H with E and F), confirming that the expanded population
seen in Lsp1/ mice by FSC/SSC analysis (as
shown in panels E and F) are macrophages.
Cumulative data of myeloid development in wt and
Lsp1
The increase in percent macrophages is accompanied by a significant
diminution of the percent small lymphocytes in the peritoneum of
Lsp1
Although we did not induce any inflammation in the mice used in this
part of our study, we considered the possibility that LSP1 We next investigated whether the increased macrophage numbers found in
the peritoneum of Lsp1
To investigate whether LSP1 deficiency affects other resident
macrophage populations, we analyzed alveolar macrophages recovered by
bronchoalveolar lavage from wt and Lsp1
Accelerated kinetics of changes in leukocyte populations in
inflamed peritoneum from Lsp1 / mice as compared to wt mice. At 8 hours total cell and macrophage numbers reached maximal values in
Lsp1/ mice, which were significantly higher
as compared to wt. Flow cytometric analysis revealed that the majority
of macrophages found in the peritoneum is
Mac-1hiCD5 in both genotypes, the ratio of
percent CD5 to CD5+ being 10.32 ± 2.25
versus 9.39 ± 1.64 in 4 wt versus 4 Lsp1/
mice. The high number of neutrophils were maintained at 8 hours in
Lsp1/ mice. At 24 hours macrophage numbers
became similar between the 2 genotypes, with wt mice reaching maximal
levels of macrophages and total cells only at this time point.
Interestingly at 24 hours, there was a dramatic decrease in the number
of LSP1 neutrophils, whereas wt neutrophil numbers
remained high. These differences between wt and
Lsp1/ mice were also reflected in total cell
numbers. No differences in the number of macrophages and neutrophils
were observed at later time points between the 2 genotypes. These data
show that during acute inflammation induced by TG, macrophage and
neutrophil recruitment and clearance of neutrophils occur more
efficiently in the absence of LSP1.
Increased chemotaxis of LSP1
TG-elicited neutrophils exhibit enhanced chemotaxis, we performed in
vitro chemotaxis assays similar to previously described assays for
mouse neutrophils42,43 (see "Materials and methods")
using the well-established chemoattractants, fMLP and the C-X-C
chemokine KC, both specific for neutrophils.45-47
Neutrophils from Lsp1/ mice display more
than 2-fold increased chemotaxis as compared to wt over a 100-fold
range of concentrations for fMLP (Figure 8). Although the response to KC was also
increased in LSP1 neutrophils this did not reach
statistical significance (P = .07). These data strongly
suggest that the increase in neutrophil numbers in
Lsp1/ mice as compared to wt at 4 hours
after TG is due to increased chemotaxis of these cells.
Normal lymphocyte development in Lsp1 / mice were
analyzed for B- and T-cell development. Cumulative data summarized in
Table 2 show that the proportion of B-cell precursors (pro- and pre-B
cells) and immature and mature B cells of the bone marrow and the
thymocyte subsets in thymus are normal in
Lsp1/ animals. In the periphery, the
proportions of immature splenic B cells and mature splenic, blood and
lymph node B and T cells from Lsp1/ mice
appear normal. These data show that LSP1 deficiency does not affect the
B- and T-cell compartments of the central and peripheral tissues.
Lsp1 / mice. Similarly, no differences were
observed in anti-OVA IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3; data
not shown). Nonimmune serum levels of IgM, IgG subclasses, and IgA were
also similar in the 2 genotypes.
Using Lsp1 In addition to an expanded peritoneal macrophage population, we
observed, in the early phase of TG injection, an increased influx of
LSP1 In view of previous findings that resident macrophages disappear
from the inflamed site within the first hour of TG injection into the
peritoneum,51 elevated LSP1 In addition to increased recruitment, we also observed a significantly
increased clearance of neutrophils at 24 hours after TG injection in
Lsp1 Our studies indicate that LSP1 does not play a major role in B- or
T-lymphocyte development. Lsp1 In conclusion, our results demonstrate that LSP1 modulates the homeostasis of leukocytes in the peritoneum and the course of acute peritonitis. The absence of LSP1 in myeloid leukocytes results in a more rapid influx of these cells and clearance of neutrophils from this compartment. Based on this functional significance and the expression pattern of LSP1, we agree with the recent suggestion63 to change the full name of LSP1 from lymphocyte-specific protein 1 to leukocyte-specific protein 1.
We thank Drs David Hackam, Ian McGilvray, and Sergio Grinstein for valuable advice; Dr Yuri Moltyaner for teaching how to harvest alveolar macrophages; Mr Andrzej Wielowieyski and Ms Vera Cherepanov for excellent technical help; and Ms Deborah Hyam and Dr Norman Iscove for their help and advice at the initial stages of this work. G.P.D. holds the Fraser Eliott Chair in Transplantation Research, V.L.M. is a recipient of a Studentship Award from the Medical Research Council of Canada and C.W. is a recipient of a Postdoctoral Fellowship Award from the University of Toronto Arthritis Centre of Excellence.
Submitted July 6, 1999; accepted May 2, 2000.
Supported by a grant from the National Cancer Institute of Canada with funds from Canadian Cancer Society (J.J.), by funds from the University of Toronto Arthritis Centre of Excellence (J.J.-B. and J.J.), and by a grant from the Medical Research Council of Canada (G.P.D.).
J.J.-B. and V.L.M. contributed equally to this report.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jenny Jongstra-Bilen, Toronto Western Hospital, Main Pavilion, Room 13-312A, 399 Bathurst St, Toronto, Ontario M5T 2S8, Canada; e-mail: jbilen{at}uhnres.utoronto.ca.
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J. Wang, H. Jiao, T. L. Stewart, M. V. H. Lyons, H. A. Shankowsky, P. G. Scott, and E. E. Tredget Accelerated wound healing in leukocyte-specific, protein 1-deficient mouse is associated with increased infiltration of leukocytes and fibrocytes J. Leukoc. Biol., December 1, 2007; 82(6): 1554 - 1563. [Abstract] [Full Text] [PDF]
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C. D. Buckley, E. A. Ross, H. M. McGettrick, Chloe. E. Osborne, O. Haworth, C. Schmutz, P. C. W. Stone, M. Salmon, N. M. Matharu, R. K. Vohra, et al. Identification of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration J. Leukoc. Biol., February 1, 2006; 79(2): 303 - 311. [Abstract] [Full Text] [PDF]
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L. Liu, D. C. Cara, J. Kaur, E. Raharjo, S. C. Mullaly, J. Jongstra-Bilen, J. Jongstra, and P. Kubes LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration J. Exp. Med., February 7, 2005; 201(3): 409 - 418. [Abstract] [Full Text] [PDF]
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C. Wang, H. Hayashi, R. Harrison, B. Chiu, J. R. Chan, H. L. Ostergaard, R. D. Inman, J. Jongstra, M. I. Cybulsky, and J. Jongstra-Bilen Modulation of Mac-1 (CD11b/CD18)-Mediated Adhesion by the Leukocyte-Specific Protein 1 Is Key to Its Role in Neutrophil Polarization and Chemotaxis J. Immunol., July 1, 2002; 169(1): 415 - 423. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
TCR, anti-CD3 (PharMingen), anti-B220 (Sigma), anti-Mac-1 (anti-CD11b, M1/70; Serotec, Oxford, UK), anti-Gr-1 (Cedarlane Laboratories,
Hornby, Ontario), anti-IgM Abs (µ-specific F(ab'2);
Jackson ImmunoResearch Laboratories, West Grove, PA), and
anti-I-Ab major histocompatibility complex (MHC) class II
(Y3P, a gift from Dr T. Watts, Toronto); biotin-conjugated mAbs
anti-F4/80 (Serotec), anti-Mac-1 (M1/70; a gift from Dr C. Guidos,
Toronto and biotinylated using sulfo-NHS-LC-biotin as described by
manufacturer, Pierce, Rockford, IL), anti-HSA (anti-CD24; PharMingen),
and anti-IgD (HB
6, a gift from Dr F. Finkelman, Cincinnati, OH and
biotinylated as above) were used in conjunction with PE- or
FITC-conjugated streptavidin (Caltag, San Francisco, CA). Isotype
control antibodies were from Caltag or PharMingen.
) in the peritoneum (Per). **, P = .0003.
/
: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1).
J Immunol.
1986;137:245-254