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Deficiency of Src Homology 2-Containing Phosphatase 1 Results in Abnormalities in Murine Neutrophil Function: Studies in Motheaten Mice¹

Joshua Kruger^*,, Jeffrey R. Butler, Vera Cherapanov^*,, Qin Dong^*,, Hedy Ginzberg^*,, Anand Govindarajan^*,, Sergio Grinstein, Katherine A. Siminovitch^,,¶ and Gregory P. Downey^2,*,

^* Division of Respirology, The Toronto General Hospital Research Institute of the University Health Network; Department of Medicine, University of Toronto; Division of Cell Biology, Research Institute, The Hospital for Sick Children; The Samuel Lunenfeld Research Institute, Mount Sinai Hospital; and ^¶ Genomic Medicine Division, University Health Network, Toronto, Ontario, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neutrophils, an essential component of the innate immune system, are regulated in part by signaling pathways involving protein tyrosine phosphorylation. While protein tyrosine kinase functions in regulating neutrophil behavior have been extensively investigated, little is known about the role for specific protein tyrosine phosphatases (PTP) in modulating neutrophil signaling cascades. A key role for Src homology 2 domain-containing phosphatase 1 (SHP-1), a PTP, in neutrophil physiology is, however, implied by the overexpansion and inappropriate activation of granulocyte populations in SHP-1-deficient motheaten (me/me) and motheaten viable (me^v/me^v) mice. To directly investigate the importance of SHP-1 to phagocytic cell function, bone marrow neutrophils were isolated from both me/me and me^v/me^v mice and examined with respect to their responses to various stimuli. The results of these studies revealed that both quiescent and activated neutrophils from motheaten mice manifested enhanced tyrosine phosphorylation of cellular proteins in the 60- to 80-kDa range relative to that detected in wild-type congenic control neutrophils. Motheaten neutrophils also demonstrated increased oxidant production, surface expression of CD18, and adhesion to protein-coated plastic. Chemotaxis, however, was severely diminished in the SHP-deficient neutrophils relative to control neutrophils, which was possibly attributable to a combination of defective deadhesion and altered actin assembly. Taken together, these results indicate a significant role for SHP-1 in modulating the tyrosine phosphorylation-dependent signaling pathways that regulate neutrophil microbicidal functions.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neutrophilic polymorphonuclear leukocytes (PMN)³ contribute to host defense primarily through destruction of pathogenic microorganisms. They fulfill this role by virtue of rapid and coordinated responses that include chemotaxis, phagocytosis, secretion of the contents of granules/vesicles, and production of reactive oxygen intermediates (1, 2, 3). These responses are initiated by the interaction of cell surface receptors with specific ligands found on microbial targets or in the inflammatory milieu that in turn elicit intracellular signaling pathways leading to biological responses (1, 2, 3). The critical importance of these signaling cascades in host defense is well illustrated by immune deficiency syndromes, such as chronic granulomatous disease (4) and deficiency of the small GTPase Rac-2 (5), in which defects in microbicidal functions render patients unusually susceptible to life-threatening infections. Paradoxically, the same effectors activated during antimicrobial responses may also engender host tissue damage, as evidenced by the inflammatory tissue damage associated with ischemia-reperfusion, sepsis, and acute lung injury (6, 7, 8). It is therefore apparent that optimization of host defense capabilities while minimizing "collateral" host tissue damage requires that PMN microbicidal responses be tightly regulated, for example, by processes that facilitate selective triggering and/or rapid cessation of such responses once the initial stimulus has been removed.

Tyrosine phosphorylation is essential in PMN responses including adhesion, chemotaxis, oxidant production, phagocytosis, and priming (9, 10, 11, 12, 13, 14, 15, 16, 17, 18). Tyrosine phosphorylation levels are, in turn, dictated by the balance of protein tyrosine kinase (PTK) and phosphatase (PTP) activity. In PMN, PTK activation can be induced by a diversity of agonists including, for example, chemotactic peptides, cytokines, and chemokines (19, 20, 21, 22). Decreases in PTP activity may also play a role in agonist-induced increases of cellular tyrosine phosphorylation. For example, stimulation of PMN with the chemoattractant fMLP or with PMA is associated with decreases in total cellular PTP activity (23, 24). Similarly, inhibition of PTP with vanadate or its peroxides has been shown to induce (25, 26) or potentiate (27) the respiratory burst, providing additional evidence that PTP modulate microbicidal responses.

While a significant contribution of PTP to the regulation of PMN functions is apparent, comparatively little is known about the role of specific phosphatases in modulating such functions. One of the transmembrane PTP expressed in PMN, CD45, has been implicated in regulation of motility (28) and Fc-mediated phagocytosis (29), but the mechanisms linking CD45 to these behaviors are not known. The Src homology 2 (SH2) domain-containing cytosolic phosphatase 1 (SHP-1) is known to be expressed in myeloid cells, including the cultured cell lines HL-60 and U937, macrophages (30, 31, 32, 33, 34), and peripheral blood PMN (35), and modulates proliferation, apoptosis, oxidant production, and adhesion in cultured U937 cells (36). These data suggest integral roles for SHP-1 in governing PMN biology. This contention is consistent with the profound myeloid defects observed in mice completely or partially deficient in SHP-1, which are denoted motheaten (me/me) and motheaten viable (me^v/me^v) mice, respectively. Both the me and me^v mutations result in defective SHP-1 RNA splicing. The me mutation is a deletion of a cytidine residue that generates a novel splice donor site in the first SH2 domain generating a frameshift with premature truncation of the mRNA that results in a null mutation. The me^v mutation is a thymidine-to-adenine transversion leading to destruction of a donor splice site that yields an in-frame insertion or deletion in the phosphatase catalytic domain resulting in an abnormal SHP-1 protein that retains 20% of wild-type activity (37). Motheaten mice exhibit an enormous overexpansion of myelomonocytic cells with consequent diffuse and progressive inflammatory tissue injury and death due to PMN/macrophage-mediated hemorrhagic pneumonitis (38, 39). The critical role for myelomonocytic cells in the genesis of this phenotype is supported by data demonstrating the capacity of anti-Mac-1 Ab treatment to markedly reduce the me/me and me^v/me^v inflammatory phenotype (40).

Identification of SHP-1 mutation as the defect responsible for the motheaten phenotype has resulted in intensive investigation of the functions of this phosphatase. Such studies have revealed that SHP-1 participates in down-regulating a broad spectrum of growth-promoting receptor-evoked activation cascades. These include, for example, the signaling pathways triggered by receptor PTK such as c-kit (41, 42) and the CSF-1 receptor (43), cytokine receptors such as the IL-3 (41) and IFN- (44) receptors, and immune receptors containing the immune receptor tyrosine-based inhibitory motif such as CD22 (45) and paired Ig-like receptor B (PIR-B) (32, 46). SHP-1 modulates these signaling cascades via a diversity of molecular interactions including dephosphorylation of receptor tyrosine kinases (41, 43, 47, 48), interaction with noncatalytic subunits of receptors (e.g., cytokine receptors), and dephosphorylation of associated Janus family tyrosine kinase (44, 49) or via interactions with cytosolic signaling effectors such as Vav, slp-76 (50), and lck (45, 51, 52, 53, 54, 55, 56, 57).

While SHP-1 functions in lymphocytes have been extensively studied, the role of this PTP in the regulation of microbicidal responses in myeloid cells is poorly understood. SHP-1-deficient bone marrow myeloid progenitor cells and macrophages demonstrated enhanced chemotactic responses to stromal cell-derived factor 1 (SDF-1), a CXC chemokine (58). SHP-1-deficient bone marrow myeloid cells (59) and macrophages (43, 60) were hyperresponsive to growth factor stimulation and adhered and spread on surfaces to a greater extent than normal macrophages (33). Interestingly, SHP-1-deficient macrophages were impaired in their ability to detach from the substratum apparently due to defective regulation of phosphatidylinositol (PI) 3-kinase (33).

In light of the apparent importance of SHP-1 in regulating macrophage function, we sought to delineate SHP-1 roles in modulating PMN behavior. To this end, we characterized the functional properties of bone marrow PMN isolated from me/me and me^v/me^v mice, which express no and catalytically inert SHP-1, respectively. As described herein, analyses of these cells revealed that SHP-1 deficiency is associated with an increase in total cellular protein tyrosine phosphorylation and a state of hyperresponsiveness as connoted by increased surface expression of the ₂ integrin, CD11b, increased adhesion to protein-coated surfaces, and increased oxidant production. Additionally, SHP-1-deficient PMN have markedly diminished chemotactic ability that may reflect impairment in deadhesion and altered cytoskeletal regulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

Percoll and Dextran T-500 were obtained from Pharmacia LKB Biotechnology (Baie D’Urfe, Quebec, Canada). Reagents for Krebs-Ringer-phosphate dextrose (KRPD) were obtained from Mallinckrodt (Paris, KY). HEPES, PMA, fMLP, N-formyl-norleucyl-phenylalanine (fNLP), cytochalasin B, scopoletin, HRP, and mouse IgG were from Sigma (St. Louis, MO). H₂O₂ was from Caledon Laboratories (Toronto, Ontario, Canada). Calcein-acetoxymethyl ester and dihydrorhodamine were from Molecular Probes (Eugene, OR). KC was from R&D Systems (Minneapolis, MN). H₂O₂ was from Caledon Laboratories.

Buffers

Na buffer contained (in mM) 140 NaCl, 4 KCl, 10 glucose, 10 HEPES, 1 MgCl₂, and 1 CaCl₂ (pH 7.4 at 37°C). KRPD contained (in mM) 120 NaCl, 4.8 KCl, 1.2 MgSO₄, 0.93 CaCl₂, 3.1 NaH₂PO₄, and 12.5 Na₂HPO₄ (pH 7.4 at 37°C).

Antibodies

A GST-fusion protein of wild-type murine SHP-1 encompassing its two SH2 domains (amino acids 1–296) was generated as previously described (37). The recombinant protein was used to generate polyclonal Abs to SHP-1 that were affinity purified and have been shown to be suitable for immunoblotting and immunoprecipitation (35, 54). A rat mAb (clone RB6-8C5) recognizing the myeloid differentiation Ag Ly-6G (previously known as Gr-1) was obtained from PharMingen Canada (Mississauga, Ontario, Canada). A mAb to SHP-1 was obtained from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine Ab 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Rat mAbs against mouse CD11b (clone M1/70) were obtained from Biosource International (Nivelles, Belgium). Rabbit anti-lactoferrin Abs were obtained from Sigma.

Motheaten mice

Mice homozygous for the motheaten (me) mutation were obtained by mating C3HeBFeJ me/+ breeding pairs. Mice homozygous for the motheaten viable (me^v) mutation were obtained by mating C57BL/6J me^v/+ breeding pairs. The breeding pairs were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained at the Samuel Lunenfeld Research Institute, Mt. Sinai Hospital (Toronto, Ontario, Canada). All mice were genotyped using PCR amplification of tail DNA as described previously (37).

Isolation of bone marrow PMN

PMN were isolated from bone marrow according to the method of Lowell and Berton (61) with minor modifications. Long bones from mice (humerus, femur, and tibia) were removed, and the ends were clipped and then flushed using a 27-gauge needle and ice-cold calcium- and magnesium-free HBSS. Clumps of marrow were broken up by repeated pipetting. Cells from the marrow were sedimented by centrifugation at 500 x g at 4°C for 5 min and resuspended in 4 ml of HBSS. The unpurified marrow was placed in a 15-ml polypropylene tube and layered over a three-step gradient (52%, 65%, and 75% Percoll diluted with HBSS). For these studies, 100% Percoll is defined as nine parts Percoll and one part 10x HBSS (Ca²⁺ free). The tube containing the Percoll gradient was centrifuged at 1500 x g for 30 min at 4°C in a swinging bucket rotor using a slow brake to prevent disruption of the layers during deceleration. The cells were then removed from the PMN-enriched fraction at the interface of the 65% and 75% layers, diluted with an equal volume of HBSS, and sedimented in a microcentrifuge for 10 s at 75% maximum speed. These cells were resuspended in 1 ml of RPMI 1640 and counted using a Coulter counter. An aliquot of the purified cells was sedimented onto a glass coverslip using a cytocentrifuge (Shandon, Pittsburgh, PA), fixed, and stained using a modified Wright-Giemsa stain (Diff-Quick, Dade Diagnostics, Aquanda, PR). Purification of bone marrow PMN from motheaten mice required a slight modification using 62% as the second step of the gradient to yield maximum purity.

Ly6G (GR-1) labeling and flow cytometry

Surface expression of Ly6G (GR-1) was quantified with purified rat mAbs (clone RB6-8C5, PharMingen) or using undiluted supernatant from the hybridoma cell line. Cells in suspension were fixed with 1.6% paraformaldehyde in PBS for 30 min, washed with PBS twice, and incubated with the purified Ab (40 µg/ml) or undiluted hybridoma supernatant for 1 h at room temperature. Cells were again washed three times with PBS and resuspended in a 1:1500 dilution of anti-rat FITC-conjugated secondary Ab for 1 h at room temperature and washed twice with PBS. The surface expression of Gr-1 was then quantified using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

SDS-PAGE and immunoblot analysis

For assessment of cellular tyrosine phosphorylation, 1 x 10⁶ cells were suspended in 0.5 ml of Na buffer at 37°C and exposed to agonists at 37°C as specified. The reaction was stopped with 1 ml of ice-cold Na buffer, and the cells were sedimented and resuspended in 50 µl of boiling Laemmli sample buffer. The cell lysates were resolved on a 10% polyacrylamide gel using SDS-PAGE, transferred to nitrocellulose, and blotted with anti-SHP-1 or 4G10 anti-phosphotyrosine Abs as indicated.

SHP-1 immunoprecipitation and phosphatase assay

Bone marrow PMN were resuspended in 1 ml ice-cold lysis buffer (PBS (ph 7.4), 1% Nonidet P-40, 1 mM PMSF, 0.5 mM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Lysates were centrifuged at 15,000 x g for 15 min, and supernatants were mixed with 10 µl polyclonal anti-SHP-1 at 4°C for 2 h and then incubated with 50 µl protein G/A plus agarose rotating overnight at 4°C. The washed beads were analyzed by SDS-PAGE and Western blotting with monoclonal-SHP-1 Abs. Tyrosine phosphatase activity was measured in anti-SHP-1 (polyclonal) immunoprecipitates using the malachite green phosphatase assay with a phosphopeptide substrate, RRLIEDAEpYAARG (Upstate Biotechnologies). The activity was normalized to the amount of immunoreactive SHP-1 protein as determined by Western blotting of the immunoprecipitates with monoclonal anti-SHP-1 Abs followed by densitometric analysis as described below. The intensity of the SHP-1 band in each sample was determined using IP Lab Gel-D10 (Scanalytics, Fairfax, VA). The latter was calibrated by running varying amounts of recombinant GST-SHP-1 fusion protein on the same blot to ensure that samples were in the linear range of the x-ray film.

Chemotaxis

Chemotaxis was determined using a micro-Boyden chamber from Neuroprobe (Cabin John, MD). Zymosan-activated mouse serum (heterologous mouse serum incubated with zymosan for 1 h at 37°C) diluted with RPMI + 1% BSA, fMLP (10^-4–10^-7 M), and KC (10^-6–10^-8 M) were used as chemoattractants. The chemotaxis assay was conducted in RPMI with 1% BSA. In this assay the cells had to pass through a 3 µm-pore-diameter filter and were then trapped with a 0.45-µm pore diameter filter (both filters were mixed esters of cellulose). Chemotaxis was allowed to proceed for 2 h at 37°C, after which time the chamber was disassembled and the trap filter removed, and cells were fixed, stained with hematoxylin, cleared with xylene, and mounted on a slide using Permount. The cells present on the trap filter were counted using transmitted light microscopy.

Chemokinesis

Glass coverslips (Fisher Scientific, Pittsburgh, PA) were coated with fibrinogen (Sigma) for 2 h at 37°C. PMN (7.5 x 10⁴ cells) were allowed to adhere to the coverslips for 10 min at 37°C. Following incubation, the coverslips were placed in a Leiden chamber on the stage of a Leica (Deerfield, IL) DM-IRB microscope, covered with HBSS containing 1% BSA, and maintained at 37°C. fMLP (final concentration 10^-5–10^-8 M) was then added and dispersed using gentle pipetting. For analysis, fields containing equal leukocyte density were chosen for observation, and differential interference contrast images at x20 magnification were acquired at 20-s intervals for a total of 20 min using a Princeton Instruments (Trenton, NJ) Pentamax cooled charge-coupled device camera controlled by MetaFluor software (Universal Imaging, Media, PA). Following acquisition, 30 adherent PMN were chosen at random from each mouse, and the movement of their centroids were tracked using Metamorph software (Universal Imaging). Only cells that remained within the field of observation for the entire experiment were chosen. Two indices of movement were derived. The total distance traveled by each cell represents the sum of the distances between the position of the cell centroid in each 30-s interval over the period of observation. The net distance represents the distance between the cell centroid at the start and the end of the observation period. The motion of cells from motheaten and wild-type congenic control mice were analyzed randomly by a blinded observer on the same day. A total of three me/me, three me^v/me^v, and three control mice were studied.

Measurement of cytoskeletal alterations

The quantity of polymerized F-actin in neutrophils was measured as previously described (62). Briefly, after exposure to chemoattractant or vehicle control, cells were simultaneously fixed and permeabilized with lysophosphatidylcholine (0.1 mg/ml final concentration) in buffered formalin. After a 5-min incubation at 37°C, nitrobenzoxadiazole-phallacidin was added to a final concentration of 1.65 x 10⁷ M. Cells were examined on a FACScan (Becton Dickinson) and were gated on the forward and right angle light scatter to remove debris and cell clumps. Cellular fluorescence was quantified using the FL1 detector (488-nm excitation and 530-nm emission wavelengths), and values are expressed as relative fluorescence index by dividing the linearized fluorescence of the experimental group by the value for the unstimulated control cells. This method has been shown to correlate with biochemical measurements of F-actin.

The distribution of F-actin within cells was examined using Alexa-Red phalloidin (Molecular Probes) essentially as previously described (63). Briefly, after exposure to chemoattractant or vehicle control, cells were fixed with 4% paraformaldehyde, washed, and allowed to settle on coverslips that were previously coated with 1 mg/ml poly-L-lysine. After 20 min, the coverslips were rinsed in PBS and the neutrophils permeabilized by incubation in 0.1% Triton X-100 in KRPD for 15 min. The cells were stained with 1.65 x 10^-7 M Alexa-Red phalloidin for 10 min at 37°C and then washed with several rinses in PBS. The coverslips were mounted with fluorescence mounting medium (Dako, Carpinteria, CA). The slides were viewed using a Leica DM-IRB inverted fluorescence microscope and digital images captured as TIFF files using a Princeton Instruments Micromax cooled charge-coupled device camera controlled by Metamorph software. The images were imported into Adobe Photoshop, labeled, and printed on a Hewlett Packard Ink Jet printer.

Measurement of cell deformability

PMN deformability was assessed by measuring the pressure needed to pass a suspension of PMN through a polycarbonate filter (Poretics, Livermore, CA) with a uniform pore diameter of 6.5 µm (range 6.0–7.0 µm; coefficient of variation <10%) as previously described (1, 2, 3, 64, 65). In brief, polycarbonate filters, polypropylene chambers, and siliconized plastic i.v. tubing were protein coated by incubation in 20% heat-inactivated murine plasma at 37°C for 2 h to minimize cell adhesion to the tubing and chambers. A multichannel infusion pump (Harvard Apparatus, Millis, MA) was used to provide a constant flow rate of the buffer across the filters. Immediately upstream of each filter chamber, a pressure transducer (Validyne Engineering, Northridge, CA) connected to a strip chart recorder continuously measured pressure. A cell suspension (0.5 x 10⁶ cells/ml) was filtered at a constant flow rate of 1 ml/min for 300 s. Where indicated, 10^-6 M fMLP was added to the cell suspension. The maximum pressure attained was recorded for each experiment.

Measurement of oxidant production

Oxidant production by murine PMN was quantified in two ways. To analyze the kinetics of oxidant production, a fluorescence assay with scopoletin (the fluorescence of which decreases in the presence of H₂O₂) was used as previously described (36, 66). Typically, 5 x 10⁵–1 x 10⁶ cells were incubated in a physiological buffer containing 200 nM scopoletin, 2.4 U/ml HRP, and 0.01% sodium azide. PMA (10^-8 M) was added to cells suspended in scopoletin assay buffer and incubated in the fluorometer with stirring at 37°C for 3–5 min. Reduction in fluorescence of scopoletin was quantified in a Hitachi F-2000 fluorescence spectrophotometer using an excitation wavelength of 365 nm and an emission wavelength of 473 nm. A continuous readout of fluorescence vs time was obtained, and the slope of this line was calculated using graphical analysis. Standard curves were generated using known amounts of H₂O_2. To calculate the lag time of oxidase activation, the time from the addition of stimulus to the intercept of the line of maximal slope of the curve on the abscissa was measured.

Oxidant production was also measured by flow cytometry using the oxidant-sensitive dye dihydrorhodamine 123 (67) as previously described (66). In brief, 5 x 10⁵ cells in suspension were incubated in the presence of 2 µM dihydrorhodamine for 20 min at 37°C. Cells were fixed with 1.5% paraformaldehyde before analysis on a FACScan flow cytometer (Becton Dickinson).

Phagocytosis

The phagocytic ability of PMN was assayed by incubating serum- or murine IgG-opsonized zymosan with cells in the presence of the permeant fluid-phase marker Lucifer Yellow as previously described (68). PMN (3 x 10⁵) were allowed to settle on glass coverslips for 30 min at room temperature. To synchronize phagocytosis, the opsonized zymosan (6 x 10⁵ particles) was added to cells and allowed to bind for 10 min at 4°C. The temperature was then rapidly raised to 37°C and phagocytosis allowed to proceed for 10 min in the presence of 2 mg/ml Lucifer Yellow. The coverslips were cooled in an ice-water bath, and phagosomes were counted using a fluorescence microscope (Nikon, Melville, NY).

Flow cytometric analysis of CD11b and lactoferrin

Purified PMN (1 x 10⁶) were fixed with 1.6% paraformaldehyde for 15 min at room temperature, washed, and then incubated with 20% goat serum for 30 min to block nonspecific binding. Cells were washed and then incubated with 10 µg/ml rat anti-murine CD11b Ab (clone M1/70; Biosource International) or rabbit anti-human lactoferrin (Sigma) for 1 h at 4°C and washed and then incubated with FITC-labeled goat anti-rat or goat-anti-rabbit Ab. Cells were washed and resuspended in PBS and analyzed by flow cytometry (FACStar, Becton Dickinson). Where indicated, cells were first permeabilized with 0.5% Triton X-100 before incubation with the primary Abs.

Adhesion assay

Purified PMN (2 x 10⁷) were labeled with 1.5 µM calcein-acetoxymethyl ester for 20 min at 37°C with gentle agitation followed by washing and resuspension in Na buffer. Subsequently, cells were added to 24-well tissue culture plates (5 x 10⁵ cells/well) precoated with FBS and incubated for an additional 2 h at 37°C. Each assay was done in quadruplicate. After incubation, cells were fixed with 1.6% paraformaldehyde for 40 min at room temperature, and then wells were washed two times with PBS using a gravity washing device. Calcein was extracted by adding methanol to the remaining adherent cells followed by vigorous pipetting. Fluorescence was detected using a Hitachi F-2000 fluorescence spectrophotometer with an excitation wavelength of 490 nm and an emission wavelength of 520 nm. All values were normalized to the number of cells added, which was determined by measuring the mean fluorescence of three separate aliquots of 5 x 10⁵ calcein-labeled cells by methanol extraction.

For experiments using blocking anti-CD11b Abs, cells were preincubated with or without blocking anti-CD11b Abs (clone M1/70, 25 µg/ml) for 20 min at 4°C and then added to 96-well plates (1 x 10⁵ cells/well) precoated with FBS. The plates were incubated for an additional 2 h at 37°C in the presence or absence of stimulant as indicated and in the presence of anti-CD11b Abs or buffer control. The cells were fixed with 1.6% paraformaldehyde for 40 min at room temperature, and then the wells were washed two times with PBS using a gravity washing device. The fluorescence of each well was then determined using a plate-reading fluorescence spectrophotometer (Cytofluor, PE Biosystems, Mississauga, Ontario, Canada). All values were normalized to the number of cells added, which was determined by measuring the mean fluorescence of three separate aliquots of 1 x 10⁵ calcein-labeled cells.

Data analysis

Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé) or by paired or unpaired Student’s t test, as indicated. Statistical significance was considered for p values of < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of myeloid leukocytes from the bone marrow of me/me and me^v/me^v mice

To investigate the roles of SHP-1 in modulating PMN, we took advantage of the availability of SHP-deficient mice in which SHP-1 protein is either absent (me/me) or catalytically impaired (me^v/me^v). Initial attempts to purify PMN from peripheral blood using discontinuous plasma-Percoll gradients (69) yielded insufficient numbers of cells for study. These cells were therefore instead purified from bone marrow using discontinuous Percoll gradient centrifugation and a modification of previously published methods (70). This strategy allowed for the isolation of 3 x 10⁶ cells per mouse that were 88–95% mature myeloid cells as determined by modified Wright-Giemsa staining (Fig. 1, c and f).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Purification of mature PMN from bone marrow of wild-type and motheaten mice. Cells from the marrow of long bones of wild-type and me/me mice were removed, washed, and saved for analysis (a and d) or subjected to further purification using a three-step Percoll gradient as described in Materials and Methods (b and c, e and f). Cells from the PMN-enriched fraction, the interface of the 65% (wild type) or 62% (motheaten) and 75% layers, were removed and washed. Surface expression of Ly6G (GR-1) on unpurified (i.e., before Percoll gradient purification; a and d) and purified (b and e) bone marrow cells from wild-type and me/me mice was determined by flow cytometry using an mAb (clone RB6-8C5) and secondary anti-rat FITC-conjugated Ab. Note that in the unpurified marrow from me/me mice (d), a larger proportion of cells have an intermediate level of Ly6G expression (population of cells delimited by the second gate). The expression of Ly6G on cells from mev/mev mice was similar to those from me/me mice (not illustrated). The Percoll gradient centrifugation yielded cells with the highest level of Ly6G expression (b and e). An aliquot of the purified Ly6Ghigh cells was sedimented onto a glass coverslip using a cytocentrifuge and stained using a modified Wright-Giemsa stain (c and f). Note the presence of ring-shaped nuclei in many of the cells, which is characteristic of mature PMN from both the marrow and the peripheral blood (not illustrated) of mice. Cells purified from the marrow of mev/mev mice were similar to those from me/me mice (not illustrated). g, Expression of immunoreactive SHP-1 protein by purified bone marrow PMN from congenic wild-type (C3H and C57BL/6), me/me, and mev/mev mice. Proteins from whole-cell lysates of bone marrow PMN purified by Percoll gradient centrifugation from wild-type, me/me, and mev/mev mice were separated by SDS-PAGE gels and subjected to immunoblotting with affinity-purified polyclonal anti-SHP-1 Abs. For each wild-type and mutant pair, equal cell equivalents were loaded onto each lane. The two immunoreactive bands of 68 and 71 kDa in cell extracts from mev/mev PMN (lane 4) likely represent splice variants of SHP-1. The Western blots illustrated are representative of three experiments with cells from separate animals.

To determine the level of expression of SHP-1 in bone marrow PMN, the purified cell populations were subjected to anti-SHP-1 immunoblotting analysis. As indicated in Fig. 1g, the results of these analyses confirmed the presence of SHP-1 in PMN from wild-type mice (lanes 1 and 3) and absence of SHP-1 in PMN from me/me mice (lane 2). Two immunoreactive bands of 68 and 71 kDa were detected in PMN from me^v/me^v mice (Fig. 1g, lane 4) as is consistent with previous data demonstrating two SHP-1 species, probably representing splice variants, in me^v/me^v bone marrow cells and human peripheral blood PMN (35). Tyrosine phosphatase activity was severely diminished in anti-SHP-1 immunoprecipitates from bone marrow PMN from me^v/me^v mice (<10% control; data not illustrated), consistent with previous reports in me^v/me^v lymphocytes (37).

Following confirmation of impaired SHP-1 PTP activity, bone marrow PMN from the various mice were compared with respect to the intensity and pattern of cellular protein tyrosine phosphorylation. As shown in Fig. 2, the results of anti-phosphotyrosine immunoblotting analysis revealed tyrosine phosphorylation of multiple polypeptides to be enhanced in me/me and me^v/me^v relative to wild-type PMN. These increases in tyrosine phosphorylation level were observed both in quiescent cells (Fig. 2, a and b, lanes 1 and 5) and in cells activated by agonists such as the chemoattractant formyl peptide fMLP (lanes 2 and 6), PMA (lanes 3 and 7) and the phagocytic stimulus opsonized zymosan (OP-Z; lanes 4 and 8). These data reveal that the loss of SHP-1 activity is associated with increases in cellular protein tyrosine phosphorylation that is likely to impact upon the function of multiple PMN signaling effectors.

View larger version (55K): [in this window] [in a new window]   FIGURE 2. PMN from motheaten mice demonstrate enhanced tyrosine phosphorylation of multiple polypeptides. Protein from whole-cell lysates of bone marrow PMN from congenic wild-type, me/me (a), and mev/mev (b) mice was separated by SDS-PAGE gels and subjected to immunoblotting with monoclonal anti-phosphotyrosine Abs. Where indicated, cells were incubated with buffer (control; C) or activating agents including the chemoattractant peptide fMLP (10-6 M), PMA (TPA; 10-7 M), or serum-opsonized zymosan (OP-Z). Equal numbers of cell equivalents were loaded onto each lane. Western blots illustrated are representative of three experiments. Exposure of cells to fNLP and to fMLP (not illustrated), respectively, gave similar results.

The microbicidal function of myeloid cells is dependent in part on their ability to produce reactive oxygen intermediates such as O₂^- and H₂O₂ by a multicomponent enzyme complex termed the NADPH oxidase (75). To determine the relevance of SHP-1 to the regulation of the NADPH oxidase, PMN from the motheaten and wild-type mice were compared with respect to their H₂O₂ production following treatment with fMLP, C5a, and PMA, the latter of which is a direct activator of protein kinase C and a potent agonist of the NAPDH oxidase. The results of this analysis revealed that agonist-induced oxidant production was markedly increased in me/me and me^v/me^v PMN when compared with cells from wild-type mice (Fig. 3a). As is evident from the figure, levels of PMN oxidant production were higher in the me/me than in the me^v/me^vmice, possibly reflecting the presence of residual SHP-1 activity in these latter cells. To analyze the kinetics of oxidant production, H₂O₂ generation was also evaluated using a continuous assay based on scopoletin reduction. As shown in Fig. 3, b and c, the results of this analysis revealed that the maximal rate of H₂O₂ generation (as indicated by the slope of the line) was greater in me/me than in wild-type PMN, while me^v/me^v PMN showed maximum rates of oxidant production that were intermediate between those of wild-type and me/me cells (not illustrated). No differences were noted between wild-type and me/me PMN either in the lag phase (an index of the assembly phase of the oxidase; wild-type 1.16 ± 0.03 min vs me/me 1.02 ± 0.02 min, n = 4, p = NS) or in the duration (>20 min for both wild-type and me/me cells) of the oxidative burst in response to PMA. These observations suggest a role for SHP-1 in suppressing the biochemical events regulating the activity of the fully assembled respiratory burst oxidase.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. PMN from motheaten mice demonstrate enhanced oxidant production in comparison with wild type. a, Oxidant production (H2O2) by bone marrow PMN from wild-type, me/me, and mev/mev mice was determined using dihydrorhodamine with analysis by flow cytometry as described in Materials and Methods. Where indicated, cells were incubated with either buffer (unstimulated) or activating agents including the chemoattractant peptide fMLP (10-6 M), the activated form of the fifth component of complement (C5a; 10-6 M), or PMA (10-7 M). Values represent the means ± SE of at least four separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé). *, Significance at p < 0.05; **, significance at p < 0.01. b, Analysis of the kinetics of oxidant production (H2O2) by bone marrow PMN from wild-type, me/me, and mev/mev mice. Oxidant production was monitored by reduction of scopoletin with analysis by fluorescence spectroscopy as described in Materials and Methods. Equal numbers of PMN from wild-type and me/me mice were stimulated with 10-7 M PMA at 37°C in buffer containing scopoletin, HRP, and sodium azide. Reduction in fluorescence of scopoletin was monitored in a fluorescence spectrophotometer (Hitachi F-2000) with excitation wavelength of 365 nm and emission wavelength of 473 nm. The conditions of this assay were designed to measure the rate rather than the total (cumulative) amount of oxidant production. These conditions were substrate limited, and the flattening of the curve in the latter part of the assay represents substrate depletion rather than termination of oxidant production. The traces are representative of four separate experiments. Responses of bone marrow PMN from mev/mev mice were similar to those from me/me mice (not illustrated). c, Graphical representation of the data from four experiments comparing the rates of PMA-stimulated oxidant production between PMN from wild-type and me/me mice. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé). *, Significance at p < 0.05; **, p < 0.01.

Adhesive interactions between leukocytes and endothelial cells play a paramount role in regulating emigration of circulating PMN from the blood into inflamed tissues (76, 77). To assess the contribution of SHP-1 to this leukocyte function, me/me and wild-type PMN were compared with respect to their adhesion to serum-coated plastic, in the presence or absence of fMLP, C5a, and PMA, enhancers of leukocyte adhesion (78, 79, 80). As shown in Fig. 4a, an initial evaluation of PMN adherence to fibrinogen-coated plastic revealed that the numbers of cells adhering and spreading on this surface were increased in me/me relative to wild-type mice. The results of a quantitative comparison of basal and stimulated adhesion of bone marrow PMN from wild-type and me/me mice (Fig. 4b) also revealed that me/me PMN were hyperadherent and indicated a relative inability of these latter cells to modulate their adhesion in response to C5a, fMLP, or PMA. PMN from me^v/me^v mice exhibited a similar degree of hyperadhesiveness, as did the me/me cells (not illustrated).

View larger version (45K): [in this window] [in a new window]   FIGURE 4. PMN from motheaten mice are hyperadherent to protein-coated plastic. a, Representative phase-contrast images of PMN from wild-type and me/me mice adherent to protein (fibrinogen)-coated plastic. Equal numbers of cells were loaded into each well of 12-well plates, incubated for 30 min at 37°C, and washed extensively using a gravity washing device. Note the increased numbers of adherent PMN from me/me mice compared with wild-type controls. b, Adhesion of PMN to protein-coated plastic was quantified using fluorescence labeling as described in Materials and Methods. Where indicated, cells were incubated for 30 min at 37°C with either buffer (control) or activating agents including the chemoattractant peptide fMLP (10-6 M), the activated form of the fifth component of complement (C5a; 10-6 M), or PMA (10-7 M). Values represent the mean ± SEM of at least four separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé); *, significance at p < 0.05. The values for me/me do not differ between quiescent and stimulated cells.

Surface expression of ₂ integrins is increased on motheaten PMN

As adhesion of myeloid leukocytes to endothelial cells and also extracellular matrix proteins is mediated primarily by ₂ integrins (81, 82), me/me and me^v/me^v and wild-type PMN were evaluated for surface expression of CD11b, the major isoform of the -chain expressed by these cells. Flow cytometric analysis of cells stained with a mAb that recognizes CD11b revealed that surface expression of this adhesion receptor was increased in both the me/me and me^v/me^v cells compared with wild-type PMN in the absence of activating stimuli. Additionally, the relative increase in surface expression of CD11b in response to agonist stimulation was less (and statistically insignificant) in motheaten compared with wild-type PMN (Fig. 5a). As is consistent with this observation, pretreatment with a blocking anti-CD11b Ab (M1/70) reduced both the basal and agonist-stimulated adhesion of motheaten cells to a level similar to that of wild-type PMN (Fig. 5b). These data therefore suggest that modulation of ₂ integrins contributes to the enhanced adhesive properties of SHP-1-deficient PMN.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Surface expression of the 2 integrin CD11b/18 is increased on motheaten mice. a, Surface expression of CD11b on purified bone marrow PMN isolated from wild-type, me/me, and mev/mev mice was quantified with a monoclonal anti-murine CD11b Ab (M1/70) using flow cytometry as described in Materials and Methods. Where indicated, cells were incubated with either buffer (unstimulated) or activating agents including the chemoattractant peptide fMLP (10-6 M), the activated form of the fifth component of complement (C5a; 10-6 M), or PMA (10-7 M) for 10 min at 37°C. Values represent the mean ± SEM of four separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé); *, significance at p < 0.05; **, significance at p < 0.01. The differences between quiescent and agonist-stimulated motheaten PMN did not reach statistical significance. b, Function-blocking Abs directed against CD11b decrease adhesion of motheaten PMN. The adhesion of bone marrow PMN isolated from wild-type, me/me, and mev/mev mice to serum-coated plastic was assayed as described in Materials and Methods in the absence or presence of blocking concentrations (25 µg/ml) of the anti-CD11b Ab (clone M1/70). Where indicated, cells were incubated for 30 min at 37°C with either buffer (control), the chemoattractant peptide fMLP (10-6 M), C5a (10-6 M), or PMA (10-7 M). Values represent the mean ± SEM of four separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé); *, significance at p < 0.05. c, Assessment of plasma membrane and intracellular stores of CD11b and lactoferrin in motheaten and wild-type PMN. The amount of cellular CD11b and lactoferrin was measured in intact and Triton X-100-permeabilized PMN from wild-type and motheaten mice by flow cytometry using rat anti-murine CD11b and rabbit anti-lactoferrin Abs, respectively. The anti-CD11b Ab was directly conjugated to FITC, and a PE-conjugated goat anti-rabbit Ab was used to detect the anti-lactoferrin Ab. Cellular fluorescence was quantified using flow cytometry. The fluorescence in intact cells reflects the plasma membrane (cell surface) pool of CD11b or lactoferrin, whereas the fluorescence in permeabilized cells reflects the total cellular stores accessible to Ab (plasma membrane and intracellular stores).

Cell motility is defective in motheaten PMN

To assess whether SHP-1 deficiency alters cell motility, we assessed chemotaxis using a Boyden-type chamber with methylcellulose/nitrocellulose filters. As illustrated in Fig. 6, the results of this analysis revealed that chemotaxis in response to zymosan-activated serum was severely diminished in me/me and me^v/me^v PMN relative to wild-type cells. This defect was also apparent in the motheaten cells when fMLP (90 ± 6% decrease at 10^-6 M) or recombinant KC (88 ± 7% decrease at 10^-6 M) was used as the chemoattractant (not illustrated).

View larger version (24K): [in this window] [in a new window]   FIGURE 6. PMN from motheaten mice have diminished chemotaxis. Chemotaxis of bone marrow PMN isolated from wild-type, me/me, and mev/mev mice was measured in a micro-Boyden chamber using a trap filter method as described in Materials and Methods. The chemoattractant was zymozan-activated murine serum. Similar responses were observed with other chemoattractants including fMLP, fNLP, and KC but were more variable. Values represent the mean ± SEM of four separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé); **, significance at p < 0.01.

View larger version (45K): [in this window] [in a new window]   FIGURE 7. Analysis of the movement of PMN from wild-type and motheaten mice using a chemokinetic assay. a and b, The locomotion of PMN from wild-type (a) and me/me (b) mice in the presence of chemoattractant (fMLP 10 -6 M) was measured using time-lapse video microscopy as described in Materials and Methods. Filled circles represent the starting point and lines represent the path of individual cells (the position of the cell centroid at 1-s increments from beginning to end of the period of observation) as determined by computer analysis (Track Objects, Metamorph, Universal Imaging). The bar indicates 10 µm. c, Quantitative analysis of the movement of bone marrow PMN from wild-type and me/me mice in the presence of chemoattractant (fMLP 10 -6 M). Data are displayed as the total distance moved and the net distance from the origin. The total distance represents the sum of the distances between the position of the cell centroid between frames (accumulated every second) over the period of observation. The distance from origin (net distance moved) represents the distance between the cell centroid at the beginning and end of the period of observation. Values represent the mean ± SEM of three separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé); *, significance at p < 0.05.

One possible contributing factor to abnormal cell motility in motheaten PMN is defective regulation of the actin cytoskeleton. To investigate this possibility, we assessed the amount of F-actin in control and motheaten PMN before and after agonist stimulation. This analysis revealed that in quiescent PMN, the amount of F-actin was 25% higher in me/me as compared with wild-type cells (Fig. 8a). At early time points (30 s) after addition of chemoattractant, the amount of F-actin remained higher in me/me than in wild-type PMN, whereas at later time points, the amount of F-actin was similar in wild-type and motheaten cells. Stimulation of both wild-type and me/me PMN with fMLP induced shape change and a rapid redistribution of F-actin (Fig. 8b).

View larger version (47K): [in this window] [in a new window]   FIGURE 8. Assessment of F-actin levels of PMN from motheaten and wild-type mice. Bone marrow PMN isolated from wild-type and me/me mice were incubated at 37°C with 10-6 M fMLP for varying times as indicated or vehicle control followed by fixation and permeabilization and staining for F-actin as described in Materials and Methods. a, Quantification of fMLP-induced changes in F-actin by nitrobenzoxadiazole-phallacidin staining using flow cytometry. Values represent the mean ± SEM of four separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé); *, significance at p < 0.05 wild-type compared to me/me. b, Visualization of alterations in F-actin distribution using Alexa-Red phalloidin. The top panels depict quiescent cells, and the bottom panels depict cells exposed to 10-6 M fMLP for 1 min. Images are representative of cells from three separate experiments using different mice. The bar indicates 5 µm.

Another possible contributing factor to reduced movement of motheaten PMN through the pores of polycarbonate filters is decreased cellular deformability. To investigate this possibility, we compared the deformability of wild-type and motheaten PMN by monitoring the pressure required to pass a cell suspension through polycarbonate filters with 6.5-µm pores. This analysis revealed that there was no difference in the pressure required to pass quiescent wild-type or me/me PMN through the filters (filtration pressure for wild-type PMN, 2.1 ± 0.2 cm H₂O, and for me/me PMN, 2.2 ± 0.3 cm H₂O). Additionally, there was no difference in the pressure required to pass fMLP-stimulated wild-type and me/me PMN through the filters (filtration pressure for wild-type PMN, 4.1 ± 0.3 cm H₂O, and for me/me PMN, 4.2 ± 0.5 cm H₂O).

SHP-1 deficiency does not impair phagocytosis

Phagocytosis of microbial pathogens represents another important microbicidal function of PMN and a prerequisite for efficient killing. To evaluate the relevance of SHP-1 to this function, me/me, me^v/me^v, and wild-type PMN were compared with respect to their capacities to internalize serum- or IgG-opsonized zymosan. The results of this analysis revealed that the phagocytic ability of motheaten cells was comparable with that of wild-type PMN with respect to serum-opsonized (Fig. 9) or IgG-opsonized (not illustrated) zymosan.

View larger version (45K): [in this window] [in a new window]   FIGURE 9. Comparison of phagocytic ability of PMN from motheaten and wild-type mice. Bone marrow PMN isolated from wild-type, me/me, and mev/mev mice were incubated for 10 min with serum-opsonized zymosan particles (ration of 20 particles/cell) at 4°C to allow binding followed by incubation for an additional 20 min at 37°C. Phagocytosis was quantitated as described in Materials and Methods. Values represent the means ± SE of four or five separate experiments. Data were analyzed by ANOVA with correction for multiple comparisons (Sheffé).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The mechanisms by which SHP-1 exerts such diverse influence on PMN functions remain to be determined. SHP-1 has been shown to bind to a plethora of signaling effectors including growth factor receptors and cytosolic signaling molecules. Examples of the latter include Grb-2; Cbl; STAT3; STAT5a; STAT5b; Shc, the p85 subunit of PI 3-kinase; Vav, the Ras-GTPase-activating protein; and p62^DOK (43, 87, 88). SHP-1 has also been recently shown to associate with a 130-kDa tyrosyl-phosphorylated species (P130) in macrophages comprising two transmembrane glycoproteins, PIR-B/p91A and the signal-regulator protein family member BIT (31). These latter proteins are hyperphosphorylated in macrophages from me^v/me^v mice and may therefore represent SHP-1 substrates (as already shown with respect to PIR-B in B cells). Our observations that there is an enhanced respiratory burst in response to agonists that act via plasma membrane receptors (fMLP and zymosan-activated serum), as well as by agents such as PMA (a direct activator of PKC) that bypass surface receptors, indicate that there are likely additional targets of SHP-1 situated downstream in the signaling pathway leading to NADPH oxidase activation. Additional studies are required to determine the extent to which SHP-1 interactions with these or other proteins account for SHP-1 effects on myeloid cell behavior.

In concert with our previous observations, the current studies indicate that SHP-1 is important in the regulation of tyrosine phosphorylation-dependent signaling pathways in myeloid leukocytes. Our previous studies suggested a role for SHP-1 in regulation of tyrosine phosphorylation in human peripheral blood PMN (35). In these cells, activation by distinct agonists led to a time-dependent decrease in the activity of SHP-1 that correlated with an increase in whole-cell tyrosine phosphorylation. Based on these observations, a prediction would be that cells deficient in SHP-1 would have enhanced levels of tyrosine phosphorylation. Indeed, increased levels of tyrosine phosphorylation of several polypeptides were observed in PMN from motheaten mice (Fig. 2). However, it is also apparent that other factors are important in the regulation of tyrosine phosphorylation in myeloid cells, because despite the absence of SHP-1, stimulation of motheaten PMN led to a further increase in levels of cellular tyrosine phosphorylation. One interpretation of these observations is that agonist-induced increases in tyrosine kinase can be effected despite deficiency in SHP-1 and that increased activity of these kinases contributes significantly to the enhanced levels of tyrosine phosporylation after agonist exposure. Additionally, in the absence of SHP-1, alternate tyrosine phosphatases could contribute to dephosphorylation of these same phosphoproteins and the activity of these phosphatases could be modulated in agonist-stimulated cells.

The hyperadhesiveness of motheaten PMN suggests that SHP-1 is also involved in regulating the adherence properties of leukocytes. Enhanced adhesiveness of these cells appears to reflect in part an effect of SHP-1 on ₂ integrin function because surface expression of the CD11b isoform of the -chain is increased in me/me PMN and the enhanced adhesion is abrogated by anti-CD11b mAbs. PMN hyperadhesiveness may represent a contributory factor in the massive myeloid cell accumulation of observed tissues of motheaten mice (37, 83, 84), particularly in view of data revealing that the inflammatory infiltration is partially ameliorated by treatment with anti-CD11b (5C6) Ab (40).

At present the mechanism(s) whereby SHP-1 influences CD11/CD18 function and cell adhesion remain unclear, as is the role of tyrosine phosphorylation in modulating ₂ integrin function (89, 90). It is noteworthy that SHP-1 associates with tyrosine phosphorylated platelet endothelial cell adhesion molecule-1 (91) and with several molecules found in adhesion complexes including paxillin, vimentin, and F-actin in CSF-1-stimulated macrophages (87), and the relevance of these interactions with SHP-1 in modulation of cell adhesion needs to be investigated. Interestingly, several other protein tyrosine phosphatases have also been implicated in the regulation of myeloid cell-cell and cell-substrate adhesion (92, 93). For example, the closely related PTP, SHP-2, appears to play an important role in ₁ integrin-mediated activation of mitogen-activated protein kinase (94), and the leukocyte tyrosine phosphatase CD45 is required for the maintenance of integrin-mediated adhesion in murine bone marrow macrophages (95). Importantly, SHP-1-deficient macrophages were reported to have defective deadhesion attributable to an increase in d-3 phospholipids as a consequence of an increase in membrane-associated PI 3-kinase activity (33). By analogy, the hyperadhesiveness and the impaired motility in motheaten PMN observed in our study might be related to aberrant PI 3-kinase regulation.

With respect to cell motility, for cells to migrate through a chemotaxis filter or along a surface, repetitive cycles of adhesion and deadhesion are required (96, 97), and if the latter is impaired, the net effect would be diminished vectorial movement. There is a precedent for the involvement of phosphatases in regulation of deadhesion and cell motility. Maxfield and colleagues reported that chemokinesis of human PMN along a vitronectin-coated surface required the calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin (96, 98). Interestingly, these experiments revealed that effective forward motion required calcium/calcineurin-dependent release of adhesion followed by internalization and recycling of these integrins to the leading edge of migrating PMN (98). Although not investigated directly, similar mechanisms might account for defective chemotaxis and chemokinesis in motheaten PMN.

The significance of the chemotactic defect observed in vitro is uncertain because in the intact animal, circulating PMN are apparently able to emigrate from the vascular space into the tissues. It is likely, however, that multiple ligands for adhesion molecules are available in vivo that enable effective leukocyte motility despite a defective ₂ integrin function. It should be noted that bone marrow myeloid progenitors and macrophages have recently been reported to have enhanced chemotactic responses to SDF-1, a CXC chemokine (58). One potential explanation for the apparent discrepancy with our observation that mature PMN exhibited diminished chemotaxis is that chemotactic signaling pathways might have unique aspects that are dependent on the particular chemoattractant (C5a, fMLP, and KC vs SDF-1).

The relative contribution of SHP-1 and other PTP to adhesion and motility requires further investigation. To date, a number of PTP have been suggested or proven to play a role in the regulation of cell migration and adhesion. The tyrosine phosphatase LAR has been localized to focal adhesions (99), and cells from mice deficient in LAR or the closely related PTP demonstrate impairment of cell migration (100, 101, 102). LAR and the tyrosine phosphatase PTPL1 appear to regulate cell motility through interactions with the Rho and Rac G proteins (103, 104). SHP-2 and PTEN have been shown to negatively regulate focal adhesion signaling by mediating FAK dephosphorylation (105, 106, 107, 108), while PTP-PEST has been shown to mediate p130Cas dephosphorylation (109, 110) and associate with paxillin (111). While most studies suggest a down-regulatory role for PTP in cell migration, PTP directly activates c-Src (112, 113) and positively regulates focal adhesion signaling pathways (114, 115).

Regulation of chemoattractant-induced actin assembly was abnormal in motheaten PMN. The primary defect was that unstimulated PMN from motheaten mice had higher levels of F-actin when compared with wild-type controls. Consequently, chemoattractant-induced increases in F-actin were proportionately less in the motheaten cells. Although the mechanisms underlying this defective cytoskeletal regulation are not known, actin-associated proteins are known to be phosphorylated on tyrosine residues (87). Abnormalities in the regulation of tyrosine phosphorylation of proteins involved in the control of actin assembly could be disrupted in motheaten cells. This defective cytoskeletal regulation could contribute to the defective cell motility observed in motheaten PMN.

Oxidant production was also increased in motheaten PMN, an observation that suggests the involvement of SHP-1 in regulating the phagocyte NADPH oxidase. This latter protein is part of a multicomponent enzyme complex that transfers a single electron from NADPH to molecular oxygen, resulting in the production of superoxide (O₂^-) (4, 75). Although the signaling pathways leading to activation of the NADPH oxidase remain to be clarified, tyrosine phosphorylation may be relevant to the process because increases in tyrosine phosphorylation correlate temporally with activation of the oxidase (11). Additionally, inhibitors of PTK block production of reactive oxygen intermediates (11, 116), and inhibition of PTP with vanadate or its peroxides has been shown to potentiate fMLP-induced superoxide production in whole cells and to activate a respiratory burst in electroporated cells (25, 26, 27). Taken together, these observations suggest that PTP negatively regulate NADPH oxidase activation and suggest that this role is mediated at least in part by SHP-1.

It is possible that the increased plasma membrane levels of CD11b and enhanced oxidant production could be a manifestation of enhanced mobilization of secondary granules that contain intracellular stores of the ₂ integrin and the flavoprotein component of the NADPH oxidase. However, this possibility seems unlikely based on our studies demonstrating that the amounts of CD11b and lactoferrin (also contained in the secondary granules) in intracellular stores was comparable between wild-type and motheaten cells.

One potential explanation for the apparent state of hyperresponsiveness of the motheaten PMN described in our studies is that the cells are primed or activated by a systemic inflammatory response induced by elevated levels of cytokines such as TNF- produced by macrophages from these mice (38, 39, 86). However, this is unlikely to fully account for the observed phenotypic changes in motheaten PMN, because myelomonocytic U937 cells in which a catalytically inactive SHP-1 was expressed exhibited a similar pattern of enhanced oxidant production, surface expression of CD11/CD18, and hyperadhesiveness when compared with mock transfectants (36). These results argue for a more direct effect of SHP-1 deficiency leading to myeloid cell hyperresponsiveness that would be phenotypically similar to that induced in cells in response to exposure to exogenous soluble priming or activating factors such as cytokines.

In conclusion, our studies demonstrate that SHP-1 plays a central role in modulating the signaling pathways that underlie PMN microbicidal function. The predominant physiological effect of this phosphatase appears to be inhibitory, as reduced SHP-1 function is associated with enhanced activity of several microbicidal responses. These observations have potentially important implications for our understanding of disorders characterized by inflammatory tissue damage such as arthritis and ischemia-reperfusion injury (6, 7, 8, 117), the pathogenesis of which involves release of leukocyte-derived cytotoxic compounds. The potential for SHP-1 to limit leukocyte activation suggests pivotal roles for SHP-1 in terminating such pathological responses and raises the possibility that reduction in activities of PTP such as SHP-1 may contribute to aberrant PMN responses in systemic inflammatory disease.

	Footnotes

 
¹ This work was supported by operating grants from the Medical Research Council of Canada (to G.P.D., S.G., and K.A.S.). G.P.D. holds the Fraser Elliott Chair in Transplantation Research at the Toronto General Hospital of the University Health Network. K.A.S. is a Research Scientist of the Arthritis Society of Canada. S.G. is a Howard Hughes International Scholar and holds the Pitblato Chair in Cell Biology at the Hospital for Sick Children.

² Address correspondence and reprint requests to Dr. Gregory P. Downey, Clinical Sciences Division, Room 6264 Medical Sciences Building, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada, M5S 1A8.

³ Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SH2, Src homology 2; SHP-1, SH2-containing phosphatase-1; SDF-1, stromal cell-derived factor 1; PIR-B, paired Ig-like receptor B; PI, phosphatidylinositol; KRPD, Krebs-Ringer-phosphate dextrose; fNLP, N-formyl-norleucyl-phenylalanine.

Received for publication March 17, 2000. Accepted for publication August 14, 2000.

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