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Neutrophil Dysfunction in Guanosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase I-Deficient Mice¹

Claudia G. Werner^2,*, Virginia Godfrey, Roland R. Arnold, Gerald L. Featherstone, Diane Bender, Jens Schlossmann^*, Matthias Schiemann, Franz Hofmann^* and Katherine B. Pryzwansky

^* Institut für Pharmakologie und Toxikologie, Technische Universität München, München, Germany; Department of Pathology and Laboratory Medicine, and Dental Research Center, Department of Diagnostic Sciences, School of Dentistry, University of North Carolina, Chapel Hill, NC 27599; and Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, München, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI^–/–) mice. We demonstrate that murine neutrophils expressed cGKI. Similar to the regulation of Ca²⁺ by cGKI in other cells, there was a cGMP-dependent decrease in Ca²⁺ transients in response to C5a in WT, but not cGKI^–/– bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI^–/– than in WT. Enhanced chemotaxis was also observed with cGKI^–/– peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI^–/– vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI^–/– than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI^–/– (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI^–/– (60%) than in WT (90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKI down-regulates Ca²⁺ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKI on the secretagogue responses are complex, depending on the granule subtype.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The neutrophil plays a critical role in immune defense. To be effective, the neutrophil is involved in a multistep process that leads it via multiple signals from the peripheral blood to sites of inflammation. These steps involve adhesion of circulating neutrophils to the vascular wall, diapedesis, and extravasation through the basement membrane toward sites of inflammation where it eliminates foreign microorganisms through phagocytosis, generation of toxic oxygen radicals, and release of microbicidal agents from granules (1, 2, 3).

Neutrophils are recruited to sites of inflammation by a variety of chemoattractants including complement factors (C5a), IL-8, arachidonic acid (AA)³ metabolites (leukotriene B₄), and bacterial peptides (fMLP) that are generated and released locally at sites of injured tissue. Chemotactic receptors couple to heterotrimeric guanine nucleotide-binding proteins (G proteins) and elicit a wide range of responses in leukocytes including chemotaxis, respiratory burst, and granule secretion (4, 5, 6, 7). C5a, IL-8, and leukotriene B₄ are classical chemoattractants, whereas fMLP functions as a secretagogue as well. Evidence is convincing that activation of chemotactic receptors increases the concentration of cytosolic calcium ([Ca²⁺]_i) and cGMP (7, 8, 9, 10). Rat and human peripheral blood neutrophils constitutively express neuronal NO synthase (11, 12); whereas, inducible NO synthase activity is only detectable during bacterial infection (13). The findings that neutrophils generate NO (14, 15, 16), a molecule that stimulates guanylyl cyclase, and express type I cGMP-dependent protein kinase (cGKI) (17) prompted new interest in the NO/cGMP signaling pathway and its role in regulating neutrophil functions. The precise function of cGMP-elevating agents and NO donors in regulating neutrophil chemotaxis, granule secretion, and the respiratory burst is controversial (8, 10, 18, 19, 20, 21). NO has diverse effects on neutrophil functions, because a concentration-dependent biphasic effect has been reported for chemotaxis and granule secretion (22, 23, 24). Interestingly, high concentrations of NO-releasing compounds and cGMP analogues inhibit chemotaxis, whereas lower concentrations facilitate this response (20, 22, 23, 24). Evidence suggests that exogenous NO or L-Arg regulates chemotaxis and granule secretion via a cGKI-dependent mechanism (9, 22, 23, 24, 25). cGKI is an established downstream target of the NO/cGMP signaling cascade. For example, granule secretion of human neutrophils stimulated with fMLP or A23187 is associated with elevated cGMP levels and the phosphorylation of vimentin by cGKI (9, 10, 25). Similarly, during neutrophil adhesion and spreading, cGMP levels are elevated, and vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, is phosphorylated by cGKI (26). Furthermore, when neutrophils are stimulated with LPS, p38 MAPK is phosphorylated upon accumulation of cGMP (27). These findings suggest that cGKI is a regulator of neutrophil functions. On the basis of studies of neutrophils and other cell types, cGKI may regulate receptor activation and signaling pathways downstream from receptor activation (see Refs.28, 29, 30, 31), which may have an impact on the cytoskeleton (9, 10, 25, 26, 32).

Part of the controversy regarding the signal transduction pathway for cGMP/cGKI in human neutrophils may be due to the reliance on pharmacological agonists and inhibitors. The specificity of these compounds has been challenged and is questionable (33, 34). Rodent neutrophils respond to chemoattractant signaling, secrete granule contents, generate a respiratory burst, generate NO, and kill microorganisms in a manner that is both qualitatively and quantitatively similar to human neutrophils (35, 36). Therefore, to gain further insight into the regulatory role of cGKI in neutrophils, we used bone marrow-derived neutrophils and extravasated neutrophils from wild-type (WT) and cGKI-deficient (cGKI^–/–) mice to investigate the impact of cGKI on neutrophil function. The results suggest that cGKI regulates mouse neutrophil chemotaxis and granule secretion, and that, as with other cell types, cGKI lowers agonist-induced increases in [Ca²⁺]_i. In addition, cGKI may dampen the respiratory burst over time. These findings strengthen the notion and confirm previous work that cGKI is an important regulator of neutrophil functions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Antibodies

The following Abs were purchased from BD Biosciences Pharmingen: FITC-rat anti-mouse CD11b (clone M1/70), FITC-anti-rat IgG2b (isotype control, clone A95-1); R-PE-rat anti-mouse TER-119/erythroid cells (Ly-76), PE-rat anti-mouse Ly-6G (Gr-1), and Ly6C (RB6-8C5); and cytochrome c rat anti-mouse CD45 (Ly-5, clone 30-F11), anti-CD16/CD32 (Mouse Fc Block, clone 2.4G2).

Animals

The generation of the cGKI-knockout (KO) mouse has been described previously (37). The cGKI^–/– mice were kept on the 129/Sv background and were further backcrossed either to BALB/c or to C57BL/6NN background for nine generations. The animals were kept under standard housing conditions (21 ± 1°C, 55 ± 10% relative humidity, 12-h dark-light cycle, pathogen free). All investigations were performed on 4- to 6-wk-old mice that were housed in standard (type 2) hanging rodent cages (Ehret) or static microisolator cages (Ehret) with food and water ad libitum. If not otherwise indicated, in most experiments 7–10 mice per group were studied per strain (129/Sv, BALB/c, C57BL/6NN). The animal experiments were conducted in accordance with the European Communities Council Directive of November 24th 1986 and were approved by the Government of Upper Bavaria, Germany. In addition, United States of America animal experiments were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the University of North Carolina’s Institutional Animal Care and Use Committee.

Isolation of peritoneal exudate neutrophils (PE-N)

Mice (strain 129/Sv and BALB/c) received injections i.p. with 0.5 ml of 3% thioglycollate. After 4 h, the peritoneal cavities were lavaged with HBSS supplemented with 0.5 mM EDTA, and the peritoneal exudate cells were collected by low-speed centrifugation. The percentage of neutrophils was determined by differential count. Briefly, cytospins were stained with Diff-Quick (Dade Behring) and identified as neutrophils, eosinophils, macrophages, and lymphocytes by standard morphology. Cell counts were obtained in five random fields, each containing 100 cells using x100 oil immersion objective. Typically, 75–80% neutrophils were recovered. The cells were resuspended in Gey’s balanced salt solution (GBSS) supplemented with 0.1% BSA and 20 mM HEPES and were used in the assays.

Bone marrow cell (BMC) preparation

BMC of mice (129/Sv and C57BL/6NN) were flushed from femurs with ice-cold HBSS supplemented with 0.1% BSA and 2 mM EDTA. The bone marrow was freed of RBC by hypotonic lysis (62 mM NH₄Cl, 20 mM NaHCO₃, and 1 mM EDTA), and the neutrophil population was positively selected by FACS using FITC anti-mouse Gr1 (BD Pharmingen). The FACS-sorted, anti-Gr1-positive leukocyte population was composed of 97% neutrophils. Cell viability was 98%, as determined by trypan blue exclusion. Greater than 99% of the neutrophils were spherical, suggesting that the neutrophils were not activated during isolation.

Isolation of platelets from peripheral blood

Cell suspensions of mouse peripheral blood were stained with PE-anti-mouse CD42d Ab (BD Pharmingen) and positively selected by FACS. The population of cells was >98% platelets.

Flow cytometry

Cell suspensions of bone marrow and peripheral blood cells (strain BALB/c) were stained as follows: PE-anti-TER 199 for detection of immature erythroid cells; cytochrome c anti-CD45 for detection of total leukocytes; FITC anti-CD11b for detection of macrophages; and PE-anti-Gr1 for detection of neutrophils. Samples were analyzed by FACS with CellQuest version 3.2.1 f1 1998 software (BD Biosciences). Peripheral blood leukocyte counts were obtained by using Beckman Coulter AcT diff with veterinary software (Beckman Coulter), and differentials were counted manually.

Western blots

Neutrophil cell pellets (C57BL/6NN) were freeze-thawed three times in liquid nitrogen and solubilized with sample buffer (0.2 M Tris, 5% SDS, 40% glycerine, 0.004% bromphenol blue, and 50 mM DTT). After SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk in TBST, and then incubated with 1/1000 rabbit anti-cGKI or 1/100 rabbit anti-cGKI for 1 h at room temperature. The membranes were washed in TBST and incubated with 1/10,000 goat anti-rabbit IgG-HRP (Dianova) for 1 h at room temperature. The wash was repeated, and cGKI and cGKI were detected by chemiluminescence (Amersham Biosciences). The polyclonal Abs specific for the isoforms cGKI and cGKI were raised against the recombinant proteins expressed in bacteria (38).

Immunofluorescence microscopy

Cytospins (129/Sv) of BMCs were fixed for immunofluorescence microscopy as described previously (10) and stained with affinity purified rabbit anti-cGKI common peptide (35-B-5-3) at 1:25 for 1 h (39). Thereafter, cells were incubated for 30 min with FITC-goat anti-rabbit IgG (Rockland). Cells were viewed on a Leitz fluorescence microscope, and immunofluorescence micrographs were acquired with an Optronics Engineering DEI-470 color video camera (Optronics International).

Chemotaxis

PE-N or BMC-derived neutrophils of 3- to 4-wk-old mice (strain 129/Sv) were used for these studies. Cells were resuspended at a concentration of 1 x 10⁶ cells/ml in GBSS/20 mM HEPES/0.1% BSA, and chemotaxis in response to C5a (1 nM to 1 µM) or IL-8 (10–1000 ng/ml) was measured using a 48-well chemotaxis chamber with a 5-µm pore size polycarbonate membrane filter (NeuroProbe). The C5a- or IL-8-stimulated cells were incubated at 37°C for 30 or 60 min, respectively. The filters were stained with Diff-Quick (Dade Behring), and the number of cells in five random fields was counted using x100 oil immersion objective.

In vivo chemotaxis

The ears of 4- to 5-wk-old BALB/c mice were painted with 10 µl of (200 mg/ml) AA in acetone (left ear) and 10 µl of acetone (right ear) to serve as a control. The animals were sacrificed after 1.5 h, and punches of each ear lobe were obtained using a sterile 5-mm AcuPunch (Acuderm). The ear punches were immediately weighed and cut into small pieces for myeloperoxidase (MPO) analysis. The remaining ear was fixed in 3.7% formalin in PBS for histology.

MPO was extracted from homogenized ear punches by suspending the material in 0.5 ml of 0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer (pH 6.0) before sonication in an ice bath for 10 s (Heat Systems; Ultrasonics). The specimens were freeze-thawed three times, after which sonication was repeated. Suspensions were centrifuged at 40,000 x g for 15 min, and 50 µl of the resulting supernatant was measured for MPO activity (see Measurement of MPO).

Superoxide assay

PE-N from BALB/c mice were used in this study. A microtiter assay measured the superoxide dismutase-inhibitable reduction of ferricytochrome c (an indirect measure of superoxide anion production) by peritoneal exudate cells (BALB/c) in response to either 100 ng/ml PMA or 1 µM fMLP in the presence of cytochalasin B (CB) as described previously (40). The change in OD₅₅₀ was monitored kinetically using a Molecular Devices V_max Kinetic Microplate Reader (Molecular Devices). The OD was recorded every 10 s following a 3 s shake. The V_max was calculated using the slope of the OD over the first 3 min for the fMLP response and through the maximum number of points before substrate exhaustion (generally 30 min) with PMA. The correlations for the curve fittings all exceeded 0.99. An extinction coefficient of 21.1 mM^–1 at 550 nm was used for ferricytochrome c, and superoxide production was expressed as nanomoles of reduced cytochrome per 10⁶ cells per min. The area under the curve (AUC) through 60 min was calculated as an estimate of the sustained production of superoxide in response to fMLP.

Granule secretion assay

PE-N were suspended at 1 x 10⁷ cell/ml in GBSS containing 0.1% BSA. Cells (40 µl) were placed in a 96-well microtiter plate (Falcon) containing 160 µl of GBSS plus 0.1% BSA in the presence or absence of DMSO (1:2000) or 10 µM CB. The plate was placed in a circulating water bath at 37°C for 5 min. The cells were then stimulated with 1 µM fMLP or 20 ng/ml PMA for 15 min. The plate was centrifuged for 10 min at 4°C at 1000 rpm, and the culture supernatant was removed and placed in tubes on ice containing 1 µl of protease inhibitor mixture. The supernatant was used to measure MPO and lysozyme activity. For total enzyme activity within a population of cells, the cells were lysed on ice with 0.1% Triton X-100 containing protease inhibitor mixture. The cells were vortexed and microfuged to remove debris. The supernatant was used to measure MPO and lysozyme activity.

Measurement of MPO

MPO activity of supernatants (50 µl) was measured in a microwell assay using 3,3',5,5'-tetramethylbenzidine (70 µl) as a peroxidase substrate (Kirkegaard & Perry Laboratories). Human MPO (Athens Research & Technology) was used as a standard. The plate was read at OD₆₅₀ after 15 min and was monitored kinetically using a Molecular Devices V_max Kinetic Microplate Reader (Molecular Devices). The standard curves were fitted using the software program InPlot (GraphPad). Data are reported as units MPO/10⁶cells released for secretion studies, and units MPO/ml for in vivo chemotaxis assays. In addition, the percentage of MPO released was calculated by dividing the amount of enzyme released by the total amount of enzyme extracted from the nonstimulated neutrophil population.

Measurement of lysozyme

Lysozyme activity of supernatants (25 µl) was measured in a microwell assay using Micrococcus lysodeikticus (125 µl) as a substrate. A standard curve was performed using egg white lysozyme. The hydrolysis of M. lysodeikticus was monitored kinetically for 1 h at OD₄₅₀ using a Molecular Devices V_max Kinetic Microplate Reader (Molecular Devices). The standard curves were fitted using the software program InPlot (GraphPad). Data are reported as micrograms of lysozyme/10⁶ cells. In addition, the percentage of lysozyme released was calculated by dividing the amount of enzyme released by the total amount of enzyme extracted from the nonstimulated neutrophil population.

Calcium measurements

Neutrophils (strain 129/Sv) were isolated from bone marrow and purified by FACS (as described above). Ca²⁺ transients were measured at room temperature in single cells loaded with the Ca²⁺ indicator fura 2-AM (1 µM). Ca²⁺ transients were elicited two times in succession by local application of C5a (0.1 µM). Cells were incubated for 5 min in the absence or presence of 8-bromo-cGMP (8-Br-cGMP) (1 µM–1 mM) before the second Ca²⁺ transient was elicited. Images were recorded with the vision 4.0 fluorescence imaging software program (T.I.L.L. Photonics), and the AUC was integrated using the software program Microcal Origin 6.0 (Microcal Software). Values were calculated by dividing the AUC of the second transient by the AUC of the first transient.

Statistical evaluations

Data are given as mean ± SEM of n experiments. Statistical significance of differences was analyzed by nonpaired or paired, two-tailed Student’s t test. A p value <0.05 was considered significant. All statistical calculations were performed with the InStat program (GraphPad).

Chemicals

HBSS and GBSS were purchased from (Invitrogen Life Technologies). Unless indicated otherwise, all other chemicals were obtained from Sigma-Aldrich or from Sigma Chemical.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Expression of cGKI in mouse neutrophils

In agreement with our early findings in human neutrophils (17), FACS-sorted murine bone marrow neutrophils (BM-N) express cGKI (Fig. 1). Immunoblot analysis revealed that mouse neutrophils express the isoform of cGKI (Fig. 1A). In contrast to murine platelets, which express both the and isoforms of cGKI, no immunoreactivity for cGKI was found in neutrophils (Fig. 1B). Neither cGKI nor cGKI was detected in neutrophils or platelets of cGKI-deficient mice.

View larger version (8K): [in this window] [in a new window]   FIGURE 1. Expression of cGKI in FACS-purified bone marrow-derived neutrophils. A and B, Western blots of cell extracts (60 µg/lane) of neutrophils and platelets from WT and cGKI–/– mice are shown for cGKI (A) and cGKI (B). Recombinant cGKI and cGKI proteins (10 ng per lane) demonstrate the specificity of the I and I Abs. The experiments were repeated at least three times with different sets of mice. The FACS-sorted cell populations of neutrophils and platelets were >95% pure.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. A, Staining of cGKI of BMCs was observed in permeabilized neutrophils (arrows) and megacaryocytes (m; control). B, No staining was observed in myeloid cells or megakaryocytes of cGKI–/– animals. C, Fine granular staining for cGKI was observed in the cytoplasm of neutrophils. The rabbit Ab used for this experiment recognized the cGKI and cGKI isoforms. Magnification, x125 for A and B and x500 for C.

Leukocyte lineages were examined in peripheral blood and bone marrow of 4-wk-old WT and cGKI^–/– littermates (Fig. 3). cGKI^–/– mice had significantly more peripheral blood leukocytes (WBC) than WT controls (13.73 ± 2.08 x 10³/µl vs 8.33 ± 0.82 x 10³/µl; n = 9; _*, p < 0.05). Interestingly, FACS analyses revealed a disproportionate increase in total numbers of neutrophils in the cGKI^–/– animals that resulted in a significant increase in the percentage of neutrophils at the expense of the lymphocytes (Fig. 3A). No significant differences in the percentage of monocytes, basophils, or eosinophils were observed between the two animal lines on blood smears stained with Wright-Giemsa or diaminobenzidine (MPO detection). In addition, all of the WBC exhibited normal morphology on blood smears. Examination of BMCs by FACS revealed no significant differences in the percentage of myeloid, lymphoid, or monocytoid lineages (Fig. 3B). Thus, the cGKI^–/– animals present with a granulocytosis in the peripheral blood.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. FACS of WT and cGKI–/– leukocytes. The percentage of WT () and cGKI–/– () gated cells by FACS is shown of peripheral blood (A) and bone marrow (B) for monocytes (MN), polymorphonuclear leukocytes (PMN), and lymphocytes (LYM). No significant differences were observed in BMCs. However, a significant increase in WBC was observed in cGKI–/– peripheral blood that resulted in a significant increase in the percentage of neutrophils. Peripheral blood, WT (n = 4), and cGKI–/– (n = 3); bone marrow, WT (n = 7), and cGKI–/– (n = 6). Four week-old WT and cGKI–/– littermate mice (BALB/c) were used. *, Significance from WT at p < 0.05.

Increased calcium transients are a hallmark of the neutrophil response to agonists (7, 41) and are modulated by cGKI in other cells (28, 42). To determine whether cGKI modulates calcium fluxes in neutrophils, we measured [Ca²⁺]_i transients in neutrophils that were stimulated with C5a. We used bone marrow-derived neutrophils to ensure that the cells were minimally activated, and chose C5a as an agonist because it induces a rise in intracellular calcium levels through a classic G protein-linked receptor.

In fura 2-loaded BM-Ns, C5a (0.1 µM) elicited [Ca²⁺]_i transients in both WT and cGKI^–/– neutrophils that reached a peak level within 1 min and returned to baseline levels within 5 min (Fig. 4A). A second exposure to C5a resulted in [Ca²⁺]_i transients of similar magnitude in neutrophils from both genotypes (Fig. 4A). In contrast, when WT neutrophils were preincubated before the second application of C5a with 8-Br-cGMP, an activator of cGKI, the size of the second transient was reduced to 68% of the first peak (Fig. 4, B and C). The reduction in [Ca²⁺]_i transients in WT neutrophils by 8-Br-cGMP was effective at concentrations as low as 1 µM, supporting the specificity of 8-Br-cGMP for cGKI. In contrast, there was no reduction in [Ca²⁺]_i induced by exogenous 8-Br-cGMP in cGKI-deficient neutrophils (Fig. 4, B and C). These data are consistent with that of other cell types (29, 37, 42). They support a cGKI-dependent regulation resulting in decreased agonist-induced Ca²⁺ transients in neutrophils.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Comparison of calcium transients in WT and cGKI–/– neutrophils. Bone marrow-derived neutrophils from WT () and cGKI–/– () mice were loaded with fura 2-AM for 45 min. [Ca2+]i transients were elicited by applying 0.1 µM C5a to adhered cells. Cells were incubated for 5 min with buffer (A) or 1 mM 8-Br-cGMP (B) before the second application of C5a. Summary of the results (C) is given as relative AUC (ratio of the second to first transient) for WT cells (control, n = 15; +8-Br-cGMP, n = 16) and cGKI–/– cells (control, n = 42; +8-Br-cGMP, n = 24). *, p < 0.02; **, p < 0.01.

In vitro chemotaxis. Chemotaxis of FACS-purified BM-Ns of WT and cGKI^–/– littermates was investigated using the prototypic chemoattractants, IL-8 and C5a. Chemotaxis of BM-Ns to IL-8 and C5a was dose dependent, with the highest chemotactic response for both WT and cGKI^–/– cells at 1 µg/ml IL-8 and 0.01 µM C5a (Fig. 5, A and B). Chemotaxis was enhanced significantly (p < 0.05) in cGKI^–/– BM-Ns compared with that of the WT littermate controls by 1 µg IL-8 and 0.1 µM C5a. There were no significant differences in random migration and chemokinesis between genotypes.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Comparison of chemotaxis of WT and cGKI–/– bone marrow-derived and peritoneal exudate neutrophils. Chemotaxis was dose dependent for IL-8 (A) and C5a (B) for WT and cGKI–/– bone marrow-derived neutrophils. Chemotaxis was significantly enhanced in cGKI–/– BM-Ns to IL-8 at 1 µg/ml (A) and to C5a at 0.1 µM (B). Similar to bone marrow-derived neutrophils, chemotaxis was dose dependent to C5a for WT and mutant PE-N (C). Chemotaxis was significantly enhanced in peritoneal exudate cGKI–/– neutrophils from 1 to 0.01 µM C5a. A, n = 3; B, n = 4; C, n = 2 WT and cGKI–/–. Incubation time was 30 min for C5a and 60 min for IL-8. All cells were obtained from litter-matched 129/Sv animals. Asterisks indicate a significant difference from WT littermate mice. *, p < 0.05; **, p < 0.01.

In vivo chemotaxis. To determine whether chemotaxis is also enhanced in vivo, we used an AA-induced inflammatory ear model. When applied topically, AA induces an acute inflammatory response involving both vascular leakage and cellular components that are highly dependent on leukotrienes (35). A significant increase in ear weight was observed in cGKI^–/– mice compared with WT controls at 1.5 h after topical AA treatment (7.89 ± 0.15 vs 4.85 ± 0.26 mg, respectively; p < 0.001) (Fig. 6A). A negligible response was observed in the vehicle control ears with no differences between the genotypes. This increase in ear weight in the cGKI^–/– mice after AA treatment indicates enhanced vascular permeability in the cGKI^–/– mice.

View larger version (7K): [in this window] [in a new window]   FIGURE 6. Acute inflammatory response to topical AA in WT and cGKI–/– mice. The ears of BALB/c WT and cGKI–/– mice were analyzed for edema (ear weight; A) and for MPO content, an index of neutrophil influx (B). Differences in weight and MPO content between AA-treated and control (acetone) ear punches were measured. A significant increase in ear weight and MPO content was observed in cGKI-deficient mice. Asterisks indicate a significant difference from WT mice ***, p < 0.001; n = 7.

View larger version (115K): [in this window] [in a new window]   FIGURE 7. Histology of ears after topical application of AA in WT and cGKI–/– mice. Histological sections of ears after topical application of acetone (control) (A and B) and AA (C and D) are shown in WT (A and C) and cGKI–/– (B and D) mice. Samples given are representative for each group of seven WT and seven cGKI–/– animals, from experiments performed on three different days. An influx of neutrophils was observed after AA treatment in both WT and cGKI–/– ears. Magnification, x40.

The effects of deleting the cGKI gene on the regulation of granule secretion and respiratory burst (below) were performed using elicited PE-N. The percentage of neutrophils in the harvested exudates was not significantly different between WT and cGKI-deficient cells. We used fMLP for these studies because it is an established activator of both granule secretion and the respiratory burst in mouse and human neutrophils, and similar to C5a and IL-8, fMLP binds to a G protein-coupled receptor for activation (43, 44). Comparative studies of fMLP with PMA, a direct activator of protein kinase C (PKC), were performed using the same population of PE-N from each animal. We used lysozyme to follow granule secretion in general, because it is packaged in primary, secondary, and tertiary granule types (45). In addition, MPO was used as a marker to measure the secretion of primary (azurophilic) granules, which are released by fMLP but not PMA (PKC-independent). We calculated the amount and the percentage of enzyme released.

For these studies, the PE-N were preincubated with CB and then stimulated with 1 µM fMLP for two reasons. First, granule secretion was not measurable in either the WT or the cGKI^–/– PE-N with fMLP in the absence of CB or with CB alone. Second, whereas actin is not a substrate for cGKI, the intermediate filament protein vimentin is phosphorylated by cGKI in human neutrophils activated with fMLP (9). We have shown that cytochalasins alter the organization of both microfilaments and intermediate filaments during granule secretion (10). Therefore, CB offers an opportunity to directly investigate the effects of deleting the cGKI gene on granule secretion independent of the cytoskeleton.

The granule content of the PE-N of cGKI^–/– animals was different from their WT littermates (Tables I and II). Although the total extractable lysozyme was similar between animals, there was more than a 2-fold increase in the total MPO extracted from the cGKI^–/–-elicited peritoneal exudate cell population compared with that of WT (p < 0.05). fMLP/CB stimulated the release of MPO and lysozyme by WT and cGKI^–/– peritoneal exudate cells. There was more than a 4-fold increase in the quantity of MPO released by cGKI^–/– peritoneal exudate cells (Table I). This increase was not simply due to the increased availability of MPO content, because there was also a significant increase in the percentage of MPO released from peritoneal exudate cells of cGKI^–/– (69 ± 10%) vs that of WT (36 ± 4%) animals. Consistent with primary granule packaging, MPO was not released in response to PMA with peritoneal exudate cells from either genotype (Table I).

cGKI^–/– neutrophils have a greater sustained production of superoxide

The effects of the deletion of the cGKI gene on the respiratory burst were performed using PE-N. As with the granule secretion studies, the PE-N were preincubated with CB and then stimulated with 1 µM fMLP. Comparative studies of fMLP with PMA were performed using the same populations of neutrophils. There is an initial response to fMLP that is sustained for 4 min, followed by a rapid decline in the rate of superoxide production. To determine the initial rate, the V_max was determined using the values in the first 3 min and the V_max software of Molecular Devices. The AUC over 60 min was calculated using the Molecular Devices software to capture the sustained production of superoxide. In contrast to fMLP, PMA activates NADPH oxidase through direct activation of PKC, resulting in a constant rate of cytochrome c reduction that is limited only by the availability of substrate. Therefore, the PMA-induced NADPH oxidase activity was estimated as a V_max using the maximum number of data points before substrate limitation. Because AUC resulting from PMA stimulation would be directly proportional to the V_max, AUC for PMA response was not a meaningful calculation. The elicited peritoneal exudate cell populations from both WT and cGKI^–/– gave similar kinetics in response to both fMLP/CB and PMA.

As with other studies, there was no measurable respiratory burst response with fMLP in the absence of CB or with CB alone (no fMLP) with either the WT or the cGKI^–/– PE-N. After fMLP/CB stimulation, there was an initial rate that was sustained for 4 min (V_maxinit) followed by a reduced rate through 60 min. The kinetics of the response from 0 to 60 min was captured by transforming the data to the AUC. As can be seen in Table III, there was a significant difference in the sustained release of O₂^– (AUC) in WT vs cGKI^–/– PE-N. In contrast, the differences in the V_maxinit values of the WT vs the cGKI^–/– PE-N responses to fMLP failed to reach significance (p = 0.29).

PMA induced a sustained rate of superoxide dismutase-inhibitable reduction of cytochrome c by peritoneal exudate cells from both WT and KO mice (Table III). The differences in responses did not reach significance (p = 0.12), suggesting that cGKI does not influence the NADPH oxidase signaling pathway downstream from PKC with extravasated neutrophils.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The regulation of neutrophil functions by cGMP/cGKI has not been adequately defined using pharmacological inhibitors, because these compounds elicit effects unrelated to cGKI (33, 34). To gain further insight into the signaling pathway of cGKI, we used neutrophils from genetically modified mice that lacked the cGKI gene. In agreement with our studies of human neutrophils, murine neutrophils express cGKI (9, 17, 25). We further demonstrated that murine neutrophils express cGKI, the isoform with the highest affinity for cGMP (31). The murine neutrophils used in this investigation were obtained from 28- to 35-day-old mice. The cGKI-KO mice die within 42 days of age, although the exact cause of death is undetermined. The mice are notably smaller than their littermates from birth until death, and have hypertension, increased platelet adhesion and aggregation, splenomegaly, intestinal dysmotility, and stenosis of the pylorus and ileo-caeco-colic valves (37, 46). There were no differences in the relative numbers of myeloid cells in the bone marrow between WT and cGKI^–/– mice. Examination of the peripheral blood showed that the cGKI^–/– mice have a mild anemia and mild reticulocytosis. In addition, there was an elevated WBC and a granulocytosis with a significant elevation of segmented and band neutrophils, suggesting inflammation. However, necropsies of the cGKI mice revealed no evidence of inflammation throughout their life span.

Similar to other cell types (28, 29, 37, 42), cGMP decreased agonist-induced Ca²⁺ transients in WT, but not cGKI^–/– bone marrow-derived neutrophils. This observation is noteworthy, because Ca²⁺ is regarded as an important second messenger in response to agonists in neutrophils (41). Thus, when cytosolic levels of cGMP are increasing, for example, during an inflammatory episode (8), cGKI may be activated by NO/cGMP signaling to phosphorylate a calcium regulatory protein that functions to lower [Ca²⁺]_i (47) and the binding subunit of myosin phosphatase (48) that results in an increased calcium sensitivity of the contractile machinery (49). In addition, modification and inhibition of the small GTPase Rho has been reported (50). The phosphorylation of these various targets may explain why moderate chelation of cytosolic calcium has been reported to have a minimal or no effect on membrane ruffling, actin polymerization, and other cytoskeletal functions (51). We conclude that deletion of cGKI can affect neutrophil motility and function by several mechanisms.

Chemotaxis

Chemotaxis to IL-8 and C5a was greater in cGKI-deficient neutrophils than WT controls, suggesting that cGKI down-regulates neutrophil chemotaxis. Both WT and cGKI-deficient PE-N were more responsive to C5a than the bone marrow-derived neutrophils, consistent with the possibility that C5a receptors were up-regulated by degranulation of secretory vesicles, gelatinase granules, and/or specific granules in both control and mutant neutrophils during transmigration to the peritoneum.

The in vitro results were confirmed in the AA ear model. Again, more neutrophils migrated into the cGKI-deficient ear than in the WT ear, as determined by MPO activity (4-fold increase) and histology. The MPO levels of control ear punch homogenates were similar in WT and cGKI^–/– mice, indicating that cGKI-deficient neutrophils migrate preferentially to sites of inflammation. Of particular relevance was the observation that extravasated cGKI-deficient neutrophils contained twice the quantity of MPO than WT peritoneal exudate controls (see secretion section below). However, even when assumptions are made that extravasated cGKI-deficient neutrophils have twice the quantity of MPO than controls, the 4-fold increase in MPO activity and the predominance of neutrophils in the ear punches over WT controls suggests that chemotaxis is greater in the cGKI^–/– animals.

Interestingly, the ear weight was significantly increased in the cGKI^–/– animals, suggesting enhanced vascular permeability in the cGKI^–/– animals. Release of eicosanoids has been shown to elevate cGMP levels in neutrophils and other cells (52, 53, 54). Activation of cGKI reduces vascular tone by a number of different mechanisms (28, 29, 48, 49). Therefore, it is conceivable that the increased tone of vascular smooth muscle in cGKI^–/– cells facilitated the increased permeability. Further studies are required to investigate cGKI signal transduction in regulating vascular permeability.

Chemotactic processes are, at least partly, dependent upon intracellular Ca²⁺. Neutrophils undergo repeated Ca²⁺ influx events as chemotaxis proceeds (55, 56, 66). It is interesting that chemotaxis is enhanced, and there is a cGMP-dependent suppression in [Ca²⁺]_i transients in C5a-stimulated BM-Ns. A correlation between the [Ca²⁺]_i and the speed of neutrophil migration has been reported (55). Therefore, it is possible that cGKI may regulate neutrophil motility by lowering agonist-induced [Ca²⁺]_i transients. In addition, cGKI may regulate neutrophil motility by affecting myosin phosphatase activity (48), thereby affecting filament formation (e.g., pseudopodia). Furthermore, VASP, a member of the actin-binding protein family Drosophila Enabled/VASP, also plays a key role in cell motility (57). In neutrophils, VASP is phosphorylated by cGKI and colocalizes with F-actin in filopodia and focal adhesions during spreading, a time when cGMP levels are elevated (26). Fibroblasts deficient in mammalian homologue of Drosophila Enabled/VASP move faster than controls (58, 59), suggesting that a possible mechanism for the increase in motility of cGKI^–/– neutrophils is the reduced phosphorylation of VASP by cGKI. It is possible that the cGKI-deficient neutrophils may move faster because agonist-evoked [Ca²⁺]_i transients remain high, thus leading to a faster turnover of focal adhesions, and that VASP and myosin phosphatase are targets regulated by cGKI (for detailed discussion, see Ref.31). Further studies are required to determine the mechanism for the abnormal chemotaxis response of cGKI-deficient neutrophils.

Granule secretion

Extravasated neutrophils were used to investigate granule secretion and the respiratory burst. Comparative studies with BM-Ns were not feasible, because large numbers of neutrophils were required for experimentation, and the survival of the cGKI-deficient animals is poor. However, in light of the fact that neutrophils perform their primary function at sites of inflammation, the PE-N offered the opportunity to investigate the responsiveness of a population of neutrophils that were recruited in vivo to an inflammatory site.

We were surprised to find that extravasated neutrophils from cGKI-deficient mice contain a significant 2-fold increase in the concentration of total extractable MPO, but not lysozyme. Therefore, this increase in MPO suggests that either there is a packaging defect for MPO during myelopoiesis, or that cGKI^–/– PE-N retained a population of MPO-containing granules that was released by WT cells en route from the peripheral blood to the peritoneum. MPO was undetectable after stimulation with PMA, a specific granule agonist, for both cGKI^–/– and WT neutrophils. If MPO packaging were defective, we might expect that MPO would be released with lysozyme after PMA stimulation. It is also possible that MPO is synthesized in greater quantity during granulogenesis, but is packaged appropriately in the primary granules. Unfortunately, we were unable to determine the MPO content of bone marrow-derived neutrophils. It is also possible that there is a subset of MPO-containing granules that is under the regulation of cGKI and is released in transit from the peripheral blood to the peritoneum. This hypothesis would predict that the WT, but not the cGKI^–/– neutrophils have released a subset of MPO-containing granules before arriving in the peritoneum.

A disorder in the release of MPO was also observed when the extravasated cGKI-deficient neutrophils were stimulated with fMLP/CB. The cGKI^–/– PE-N released more MPO than WT cells, and there was a 2-fold increase in the percentage of MPO released after fMLP/CB stimulation by cGKI-deficient PE-N vs WT controls. Interestingly, both WT and cGKI-deficient PE-N retained the same amount of MPO after stimulation with fMLP/CB (0.522 U for WT vs 0.557 U for cGKI^–/–), suggesting that both WT and mutant neutrophils may be retaining the same population of MPO-containing granules after stimulation with fMLP/CB. The defensin-rich population of azurophil granules may be retained because their primary function is to kill bacteria during phagocytosis (60). Whether or not this subset of granules is the defensin-rich population remains to be investigated. The 69% release of MPO by mutant PE-N vs 36% MPO by WT cells may represent the MPO from a granule subset(s) that was released by WT cells in transit plus the remainder MPO from other granule subsets. Thus, as neutrophils transit from the peripheral blood to the peritoneum, cGKI regulates the secretion of subsets of MPO-containing granules that are activated in transit (e.g., via adhesion receptors), and activated in the peritoneum (e.g., via inflammatory mediators such as fMLP).

By comparing the responsiveness of the same population of neutrophils that were stimulated with fMLP/CB (PKC independent and dependent) and PMA (PKC dependent), we were able to determine whether there was interdependence between cGKI and PKC signaling. Similar to WT PE-N, MPO was not released by cGKI^–/– cells after stimulation with PMA. Thus, secretion of MPO-containing granules is PKC independent, and cGKI is not involved in regulating MPO release downstream from PKC. However, after stimulation with fMLP/CB, both the quantity and percentage of MPO released by cGKI^–/– PE-N was significantly greater when compared with WT controls. Based upon the fact that MPO is not released by WT and cGKI^–/– PE-N by PMA, we suggest that the regulation of these MPO granule subset(s) by cGKI in response to fMLP/CB is PKC independent.

Unlike MPO, there were no significant differences in lysozyme content in extravasated cGKI-deficient neutrophils. Although the differences in the amounts of lysozyme secreted after fMLP or PMA were not statistically significant, the percentages of enzyme released by WT and mutant PE-N were significantly different. In WT PE-N, 88% lysozyme was released after fMLP/CB or PMA, whereas only 62% lysozyme was released by cGKI^–/– neutrophils with both agonists. These data suggest that 26% of the lysozyme-containing granule population is regulated by cGKI, and that these are specific granules (lack MPO) and are PKC dependent. We propose that the site of cGKI regulation for this subset of specific granules is downstream from PKC activation, because 26% of the lysozyme was retained in cGKI-deficient neutrophils that were stimulated with both fMLP/CB and PMA.

Both fMLP and PMA stimulate significant elevations of cGMP levels in neutrophils (10, 25, 52). We expect that cGMP levels would also be elevated in mouse neutrophils, because we and others have shown that the regulation of cGMP levels is not altered in cGKI-deficient mice (61, 62). Although the substrates for cGKI are not clear, cGKI appears to play an important role in regulating signal transduction pathways in neutrophils, particularly calcium mobilization and p38 MAPK. Calcium mobilization in and of itself induces granule exocytosis in neutrophils, and a hierarchical mobilization of neutrophil granule subsets can be achieved in vitro by gradual elevations in the intracellular Ca²⁺ levels (63). It is well known that the MPO-containing granules require more Ca²⁺ for release than the gelatinase and specific granules (60). Therefore, it is possible that [Ca²⁺]_i remain higher after fMLP stimulation in cGKI-deficient neutrophils vs WT cells, and that these elevated calcium levels promote the enhanced secretion of MPO-containing granules. In support of this hypothesis, fMLP-induced Ca²⁺ transients were inhibited in rat neutrophils by YC-1, an activator of soluble guanylyl cyclase (64). Because fMLP, like C5a, is activated by G protein coupling and evokes calcium transients, it is possible that cGKI down-regulates [Ca²⁺]_i in fMLP-stimulated neutrophils as well.

Another of the many sites of cGKI regulation may include the MAPK pathway (65), because p38 MAPK is a known substrate for cGKI in LPS-primed neutrophils that are stimulated with fMLP (27). Furthermore, there is evidence that secretion of azurophil and specific granules after fMLP stimulation is mediated by p38 MAPK that is activated via Src family tyrosine kinases (67).

Respiratory burst

Although the differences in the V_maxinit responses of the WT vs the cGKI^–/– PE-N responses to fMLP/CB failed to reach significance, measurements of the AUC revealed statistically significant differences between genotypes. The cGKI^–/– PE-N appeared to take longer to shut down production of superoxide than did WT cells, suggesting that cGKI may serve to dampen the second phase of the respiratory burst evoked by fMLP/CB. When the same cell populations were stimulated with PMA, the differences between the WT and cGKI^–/– PE-N in O₂^– production did not reach significance. These data suggest that cGKI does not affect the NADPH oxidase signaling pathway downstream from PKC with extravasated neutrophils.

In contrast to the activation of PKC by PMA, the activation of formyl peptide receptors generate multiple second messengers resulting in an increase in 1,2-diacylglycerol and inositol 1,4,5-triphosphate as well as an increase in [Ca²⁺]_i. In contrast to the PMA-induced respiratory burst, these multiple pathways evoked by fMLP stimulation result in a complex kinetics of respiratory burst, suggesting that cGKI influences the production of superoxide over time, possibly by modulation of the Ca²⁺-regulating protein (31, 47).

In summary, the neutrophil is a complex cell with multiple functions, many of which are dependent upon the selective release of cytoplasmic granules as the neutrophil progresses from a quiescent cell in the bone marrow and peripheral blood, to a migratory cell as it leaves the circulation, and finally to an inflammatory cell in the extravascular tissue. Our findings suggest that cGKI down-regulates chemotaxis and agonist-induced increases in [Ca²⁺]_i, and that it may dampen the respiratory burst over time in extravasated neutrophils. cGKI may regulate the release of a subset(s) of azurophil granules during transmigration and at sites of inflammation. Furthermore, extravasated neutrophils contain a subset of specific granules that appear dependent on cGKI for release. Further studies are required to resolve the specific sites of cGKI regulation in neutrophils.

	Acknowledgments

 
We thank Dr. Margrith Verghese for her technical advice and helpful review of the manuscript, and Pam McElveen, Sanjeda Sultana, and Astrid Lauxen for their skillful technical assistance.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from Bundesministerium für Bildung und Forschung, Deutsche Forschungsgemeinschaft, and North Atlantic Treaty Organization.

² Address correspondence and reprint requests to Dr. Claudia G. Werner at the current address: Dipartimento di Farmacologia Preclinica e Clinica, Viale G. Pieraccini, 6, 50139 Firenze, Italia. E-mail address: pharma{at}ipt.med.tu-muenchen.de

³ Abbreviations used in this paper: AA, arachidonic acid; [Ca²⁺]_i, cytosolic calcium concentration; cGKI, type I cGMP-dependent protein kinase; VASP, vasodilator-stimulated phosphoprotein; WT, wild type; KO, knockout; PE-N, peritoneal exudate neutrophils; GBSS, Geys’ balanced salt solution; BMC, bone marrow cell; MPO, myeloperoxidase; CB, cytochalasin B; AUC, area under curve; 8-Br-cGMP, 8-bromo-cGMP; BM-N, bone marrow neutrophil; PKC, protein kinase C.

Received for publication May 28, 2004. Accepted for publication May 17, 2005.

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