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Compartmentalization of Cyclic GMP-Dependent Protein Kinase in Formyl-Peptide Stimulated Neutrophils By Katherine B. Pryzwansky, Todd A. Wyatt, Harriette Nichols, and Thomas M. Lincoln The presence and physiologic role of cyclic GMP-dependent protein kinase (G-kinase) in human neutrophils was investigated by Western blot analysis and immunocytochemistry. Small quantities of G-kinase were found in the cytoskeletal-enriched fraction of neutrophil lysates as detected by Western blots using a polyclonal antibody raised against bovine aorta G-kinase. Immunofluorescencemicroscopy demonstrated in adherent neutrophils that G-kinase was localized diffusely within the cytoplasm, at the microtubule organizing center, and in the euchromatin of the nucleus. Because cyclic GMP is implicated as a modulator of neutrophil chemotaxis. G-kinase localizationwas investigated in neutrophils activated with N-formyl-methionylleucyl-phenylalanine (fMLP). fMLP stimulated transient focal changes in G-kinase localization that coincided with transient changes in cell shape. G-kinase translocated over a period of 5 minutes from diffuse staining of the cytosol to filaments within the uropod of polarizedcells (1 minute), to bundles of filaments associated with loss of cell polarity (2.5 minutes), and finally to more intense staining of the nuclear euchromatin (5 minutes). Optical sectioning of neutrophils by confocal laser scanning microscopy confirmed that G-kinase was restricted to specific sub-cellular compartments during cell activation. This transient localization of G-kinase was disrupted by cytoskeletal inhibitors and was augmented by 8-Br-cyclic GMP. These data provide evidence for the first time that G-kinase plays a physiologic role in human neutrophils, and support the concept of compartmentalization of cyclic nucleotides during neutrophil activation. 0 1990by The American Society of Hematology. C ubiquitous nor abundant, and the physiologic role of cyclic G M P is not understood. While G-kinase and its counterpart cyclic AMP-dependent protein kinase (A-kinase) are homologous proteins, the more restrictive distribution and substrate specificity of G-kinase suggest a specialized function for this At this time, G-kinase has not been isolated, characterized, or localized in neutrophils. In this report we demonstrate the presence of G-kinase in human neutrophils, and describe a transient redistribution of G-kinase to specific targeting proteins or structures in cells stimulated with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). YCLIC G M P has been implicated as a modulator of neutrophil chemotaxis because agents that elevate cyclic G M P levels enhance chemotaxis.’ Nevertheless, the physiologic role of cyclic G M P has remained an enigma for the past 15 years, since elevation of cyclic G M P levels by chemoattractants has led to conflicting A new interest in cyclic G M P has recently evolved, in that nitric oxide (NO), a potent stimulant of guanylate cyclase, is generated by neutrophils that are stimulated with chemoattractants4 Furthermore, chemotaxis is inhibited by blocking N O synthesis and is restored with either di-butryl cyclic G M P or with L-arginine, the precursor for NO prod~ction.~ The mechanism by which the rise in cyclic G M P after activation of guanylate cyclase mediates its effects in neutrophils remains to be established. An important effector for transducing cyclic G M P signals into biologic responses is cyclic GMP-dependent protein kinase (G-kinase). Although G-kinase has been characterized biochemically, its physiologic role is only now emerging, particularly in vascular smooth muscle function.6 Information on the role of G-kinase in other cells is minimal since this enzyme is neither From the Department of Pathology, University of North Carolina, Chapel Hill; and Department of Pharmacology, University of South Alabama, Mobile. Submitted February 2,1990; accepted April 13.1990. Supported by the American Cancer Society (ACS-401),National Institutes of Health (CA-07017 and HL34646). and USPHS General Research Support Award (5-501-FR-05406). T.M.L. is an Established Investigator for the American Heart Association. A portion of this report was presented at the FASEB Meetings. 3:A1086.1989. Address reprint requests to Katherine B. Pryrwansky, PhD. Department of Pathology, Brinkhous-Bullitt Building 7525. University of North Carolina, Chapel Hill. NC 27599-7525. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1990 by The American Society of Hematology. 0006-4971/90/7603-0019$3.00/0 612 MATERIALS AND METHODS Neutrophil isolation. Neutrophils were isolated from human peripheral blood with Polyresolving media (Flow Lab, McLean, VA). The cells were resuspended at 3 x lo6 cells/mL in Gey’s Balanced Salts buffered with 10 mmol/L HEPES, pH 7.2, and supplemented with 1.5 mmol/L CaCl,, 1 mmol/L MgCl,, 0.3 mmol/L MgSO, (GBS), and 10% human type AB serum. Neutrophils were allowed to adhere to glass coverslips for 15 minutes at 37OC. Unattached cells were removed by washing with GBS. Cells consisted of greater than 95% neutrophils, and were greater than 98% viable by trypan blue exclusion. All reagents were checked for endotoxin by the limulus test and contained less than 0.07 ng/mL of endotoxin. Neutrophil stimulation. Neutrophil monolayers were stimulated with lo-’ mol/L fMLP (Sigma, St Louis, MO) in GBS plus 10% human type AB serum from 30 seconds to 5 minutes at 37OC. The culture media was removed and the cells were quickly fixed in 1% paraformaldehyde for 10 minutes, followed by 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 minutes, -20°C methanol for 4 minutes, and -2OOC acetone for 1 minute. Cells were washed in PBS after formaldehyde and acetone.* In some instances cells were pre-incubated with 1 pmol/L 8-Br-cyclic GMP (Sigma) for 15 minutes, followed by incubation with lo-’ mol/L fMLP in the presence of 8-Br-cyclic GMP. Cells were also pre-incubated with 5 pg/mL cytochalasin B (Sigma) for 5 minutes or 2.5 pg/mL nocodazol (Janssen, Piscataway, NJ) for 10 minutes to inhibit microfilaments or microtubules, respectively. Immunofluorescence microscopy. Fixed monolayers were incubated at 4°C overnight with rabbit anti-bovine aorta G-kinase, B l ~ d VOI , 76, NO 3 (August 1). 1990: pp 6 12-6 18 OKINASE COMPARTMENTALIZATION IN NEUTROPHLS washed in PBS. and stained for 30 minutes with F I X goat anti-rabbit immunoglobulin G (1s)(Cappel-Worthington. West Chester. PA). The antibody to bovine aorta G-kinase has been characterized for specificity in smooth muscle cells.' Cells were mounted in polyvinyl-alcohol and viewed on a Leitr fluorescence microscope or a Zciss Confocal Dual Laser Scanning Microscope (CLSM). Immunofluorescence photomicrographs were taken on a Leitz Orthomat Camera with Kodak Tri-X Pan film, ASA 400. ConfMol-laser-sconning immunofluorescence micrarcopy. To tesolve differential staining patterns at various levels within a cell, neutrophils were optically Kctioned at 0.4 pm using a Zciss CLSM. An argon laser at 488 nm was used for excitation with a 10118p a s filter cutoff at 520 nm. using a 63X Zciss planapochromat objective lens with a numerical aperture of 1.4. This technology allows for immunofluorescence detection of specific regions of a cell without fluorescence interference from areas removed from the selccted plane of focus. The digital image was recorded on Kodak Technical Pan film 2415 using a Polaroid Freeze Frame Video Recorder. Wesfern blot unalysis. A neutrophil pellet consisting of 5 x IO' cells was lysad for 5 minutes at 25OC in 3 mL of 0.5%-Triton X-100 in 0.1 mol/L NaCI. 0.3 mol/L suctose. 3 mmol/L MgCI,. 0.01 mol/L piperazine aham sulfonic acid (PIPES). 1.2 mmol/L paminokntamidine. and 1 pg/mL Apmtinin.'" The DNA and RNA were digested with 2 mg/mL DNase I and IO mg/mL RNase A (Sigma) for I5 minutes at 25%. and (NH,),SO, was added to a final concentration of 0.1 mol/L to the lysate for 5 minutes at 25%. The lysate was centrifuged at lO.OOOg for IO minutes at 4%. and the pellet dissolved with 1% Triton X-100 in PBS. The protein was precipitated at 4% with 10% trichloroacetic acid as described.' Immunoprecipitation was performed on recovered protein ( 9 mg) using 5 pL affinity-purified rabbit anti4-kinase for 4 days at 4% followed by I O pL Protein A Sephanac (Sigma) for 4 hours at 4%. The precipitate was washed and applied to a 12% polyacrylamide vertical mini-gel and transferred to a nylon transfer membrane (ICN. Irvine. CA) as described." Transfers w m probed with rabbit anti-G-kinase followed by '"I Protein A (ICN). and exposed to X-ray film for 24 hours at -70%: RESULTS Demonsrrarion of Gkinase expression by neutrophils. Western blot analysis of neutrophil extracts showed the presence of G-kinase in human neutrophils (Fig I ) . The neutrophil antigen was a single polypeptide that comigrated with bovine aorta G-kinase on sodium dodecyl sulfatepolyacrylamide reducing gels at 77 Kd. Based on the number of cells required to detect the enzyme ( 5 x IO' cells), it is estimated that the level of G-kinase in neutrophils is approx- 1 2 Fb1. wmrnbbtuw)yo b o f ~ i n h u r w n philr. I . "1. bovirw lung M i 0 GMP-d.p.nd.nt protoin kino00 nandord I6 ng a( .nynwl. MC nor bando or. known protootytk f r a g n " Ot G - k i M d Ian. 2. noutrophll protoin o x t r m a0 doocribod in M o t r i alr and Mothod.. 613 imately 100-fold lower than that in vascular smooth muscle? Nevertheless. the kinase was enriched in the cytoskeleton fraction, suggesting that most of the G-kinase in neutrophils is localized in this compartment. In addition, cross-reactivity of the antibody, which was raised against bovine aorta G-kinase and characterized for specificity in smooth muscle cells. suggests homology (if not identity) with neutrophil G-kinase.' halizarion of Gkinase in jMLP-srimulared neurrcr phils. Immunofluorescence microscopy showed that G-kinase is transiently compartmentalized in fMLP-stimulated neutrophils. In untreated cells. G-kinase was diffusely localized throughout the cytoplasm. Staining of the microtubule organizing center (MTOC) and euchromatin of the nucleus was also observed (Fig 2A). In unstimulated cells that were polarized (30% to 50%. depending on the donor). some diffuse staining was also observed at the trailing end. or uropod. FMLP stimulated transient focal changes in G-kinase localitation that coincided with transient changes in cell shape. Within 30 seconds intense focal staining of G-kinase was observed along the cell margin and at the trailing end of polarized cells (Fig 2B). After I minute, when most of the neutrophils were polarized. G-kinase was focally localized on filaments in the uropod, and at the MTOC.No staining was observed on filaments in the uropod of polarized neutrophils that were not stimulated with M L P (Fig 2A). suggesting that fMLPstimulation is required for translocating G-kinase to specialized structures or targeting proteins in the uropod. After I minute, some staining of the nucleus was also observed (Fig 2C). By 2.5 minutes, bundles of filaments stained for G-kinase within one region of the cell. as cells displayed less polarity and became round (Fig 2D). These filament bundles were strongly reactive for G-kinase and were frequently localized adjacent to the nucleus. After 5 minutes cells were round, and cytoplasmic staining for G-kinase was. in general, less intense and confined primarily to the MTOC. However, more pronounced staining of the nuclear euchromatin was alsoobserved at this time (Fig 2E). Insignificant staining was observed in unstimulated and fMLP-stimulated neutrophils that were stained with pnimmune serum (Fig 2F). To confirm that G-kinase is focally localized at I minute, neutrophils were optically sectioned at 0.4-pm increments by C U M . The three optical sections shown in Fig 3 demonstrate that G-kinase is confined to the uropod and indentation of the nucleus, or hof. When cells were allowed to adhere to glass in the presence of 10% human serum. washed free of serum. and stimulated with MLP. similar translocation of G-kinase was observed. However. if cells were adhered in the absence of 10% human serum. cells avidly adhered to the coverslips. remained ~ well-spread throughout the time course of M L P stimulation ( 5 minutes), and did not undergo shape changes or display changes in G-kinase localitation. Staining of these cells was confined to the MTOC and euchromatin. with minimal staining observed in the cytoplasm (not shown). When serum was replaced with 1% human serum albumin (lipid-fm) or 10% heat-inactivated human serum (serum heated at 56% 614 b I for 30 minutes). cells remained spherical throughout the time course of fMLP treatment. and staining of G-kinase was difTuse (not shown). These results suggest that a heat labile serum factor(s) is required during cell adhesion to observe translocation of G-kinase and the changes in cell shape required for motility (eg. establishing polarity) after M L P stimulation. Eflecr of cyroskclcrol inhibirors. Because Western blots and immunofluorescence microscopy suggested an association of G-kinase with the cytoskeleton. cells were exposed to cytoskeletal inhibitors to determine if G-kinase localization was dependent on an intact cytoskeleton. Cytochalasin B inhibited both cell polaritation and transient changes in G-kinase localization. suggesting a relationship between microfilaments and the putative translocating or targeting protein (Fig 4A). Nocodazol. a microtubule inhibitor, did not inhibit polarization, but did inhibit the selective staining of filaments in the uropod (Fig 4B). Thus, agents that disrupt microfilaments and microtubules alter the focal staining of G-kinase on filaments. Eflect of exogenous cyc/ic GMP. G-kinase exists in an inactive state when cyclic GMP levels are not elevated. Elevation of cyclic GMP via M L P may enhance G-kinase -2. - ~ l i Z O t h o t ~ k , r m C trophih .tkmJ.1.d with fMLP tor various timos (A through D). Shown h c)-kiMSO kulbtlor, in unnknu)n.d neutrophils (A) and noutrophilr stimuktd with fMLP for 30 soconds (E): 1 minuto (C): 2.6 minuns (0): and 6 &uta (E). T r r t d c d b stminod with pro-immuno sorum ar. shown in (F) (origkul "tion x 1.600). localization in neutrophils. To test whether cyclic GMP augments the transient localization of G-kinase, cells were pre-incubated with 8-Br-cyclic GMP and stimulated with fMLP. After incubation with 8-Br-cyclicGMPalone,CUM demonstrated intense staining of the cytoplasm and some nuclear staining. However, G-kinase was also focally localized within one optical section at the MTOC (Fig SD). Enhanced changes in staining intensity and localization of G-kinase were observed in cells that were pre-incubated with 8-Br-cyclic GMP and stimulated with fMLP. Within 30 seconds. compartmentalization of G-kinase was observed at the MTOC and uropod (Fig 6). and by 2.5 minutes C U M showed that G-kinase was predominantly localized within the euchromatin of the nucleus and MTOC (Fig 7). Thus, increased levels of cyclic GMP within specific compartments may augment the transient localitation of G-kinase after chemotactic stimulation. DISCUSSION This study provides evidence for a physiologic role for G-kinase in human neutrophils. The data suggest that regulation of neutrophil activation via cyclic GMP and G-kinase is a localized intracellular event. fMLP induces G-KINASE COMPARTMENTALIZATION IN NEUTROPHILS 615 Fig 3. Localization of G-kinase in neutrophils stimulated for 1 minute with fMLP and viewed by CLSM. Neutrophils were optically sectioned a t 0.4-pm increments (B through D). Differential interference microscopy is shown in (A). G-kinase is focally localized within the nuclear hof and uropod (original magnification x 1.100). remarkable changes in G-kinase localization that are transient, and apparently cytoskeleton-dependent. Because fMLP may also elevate cyclic G M P levels through NO production: it may be predicted that 8-Br-cyclic G M P would augment the localization produced by fMLP. This is, in fact, what was observed (Figs 5 through 7). The focal localization of G-kinase during neutrophil activation together with the observed low levels of G-kinase, suggest that compartmentalized levels of this enzyme are important for the physiologic regulation of neutrophil function. Thus, the overall levels of cyclic G M P or G-kinase activity may not be as important in contributing to a biologic effect as the placement of G-kinase via targeting proteins in the vicinity of substrate protein(s). Unraveling the regulatory role of cyclic nucleotides in neutrophils is particularly difficult to resolve, because cell activation is transient, and the sites of activation are probably confined to specialized intracellular compartments or structures. Immunocytochemistry combined with CLSM was used to localize G-kinase in neutrophils that were stimulated with the chemotactic peptide fMLP. Optically sectioning cells by CLSM was particularly useful in visualizing targeting structures and/or compartments of G-kinase. With this approach, changes in localization of G-kinase during neutrophil activation suggest targeting proteins that may be important in the actions of G-kinase, and offer clues that may eventually determine the regulatory role of this enzyme within single cells. G-kinase was localized at all time points at the MTOC and nuclear euchromatin. fMLP stimulated transient focal changes in G-kinase localization that coincided with the transient changes in cell shape. A filamentous staining pattern was observed near the cell margin and within the uropod of polarized cells. When the cells began to lose polarity and become round, strong staining of filament Fig 4. Localization of G-kinase in neutrophils that were pre-incubated with cytochalasin B (A) or nocodazol (B) and stimulated with fMLP for 2.5 minutes. CytochalasinB inhibited polarization of neutrophils and focal staining of G-kinase. Diffuse staining of the uropod was observed in nocodazol treated cells (original magnification x 1.600). 616 PRYZWANSKY ET Al Fig 6. Suios of throo C U M hugon (B through DI of nwtrophih incubotod w+th 1 pnolll. RBrq d i c GMP for 16 minutn ond noinod for Gkinoao. Difforontiol intorformco m r o n is shown in (A). Cytopbomk and n w l u r noining h obsowod in most of tho sconrwd inug... At o w optic01 @on0 (D), &kill000 is diotinctfy h l h d O t tho MTOC (origin01mognitlcotion x 1,OOO). bundles was observed within one region of the cell near the nucleus. The staining intensity of G-kinase and change in cell shape were enhanced when cells were pre-incubated with 8-Br-cyclicGMP. These results suggest that increased levels of cyclic GMP augment the transient localization of G-kinase after chemotactic stimulation. It is important to note that in the absence of 10% human serum. neutrophils were nonmotile. They did not polarize and remained well-spread throughout the time course of fMLP treatment. Under these conditions, G-kinase was intensely localized at all time points at the MTOC and nuclear euchromatin, and minimally locali7A within the cytoplasm. In addition, when serum was replaced with albumin or heat-inactivated serum, cells were nonmotile after M L P treatment. as demonstrated by no changes in cell shape (polarization). Also. under these conditions no translocation of G-kinase was observed. These observations suggest that the motile activity and/or degree of adhesion to the Fig 6. L o a ( h n k n of Gkhwoo In m m o p h b thn -0 proincubmod with R B r y d i c GMP for 16 hutn ond nfmu(n.d whh fMtP for 30 ..cond..cOmp.rtnwnnliL.tbn of Gkinoao io o b d at tho MTOC ond uropod (original nugnitlcotion x 1,200). substratum may influence cyclic GMP and G-kinase activation. Keller et all?." have shown that chemokinesis is controlled by mechanisms that influence motility and adhesion, and chemotactic factors for neutrophils in human serum have been described." Studies are in progress to determine if a serum factor (eg, C5a) is required for priming G-kinase activation in neutrophils, and/or if an adhesive protein is important. The filamentous staining patterns were disrupted with cytochalasin B and nocodaml, suggesting that one or more cytoskeletal proteins are targeting proteins for G-kinase. Possible targeting proteins include cytoskeletal-associated proteins that contribute to the contractile. metabolic, and structural activities associated with cell motility. Once localized to the appropriate substrate. G-kinase might regulate the number of microtubule nucleating sites during neutrophil activation, since increased levels of cyclic GMP are associated with an increase in microtubule numbers." and INLP stimulates an increase in microtubule numbers and length.I6 Alternatively, G-kinase may mediate its effects by lowering Ca" concentrations once localized within a specific cellular compartment(s). For example, in vascular smooth muscle, cyclic G M P lowers Ca?' concentrations resulting in relaxation." G-kinase apparently mediates this efTcct. since introduction of the enzyme into cells deficient in G-kinase results in the lowering of Ca" and dephosphorylation of the light chain of smooth muscle myosin?'" The significance of G-kinase localization within the nucleus is unknown, although it is known that G-kinase phosphorylates nuclear chromatin and nuclear histones?' Recently, Schmidt et al' have demonstrated that fMLP produces a transient increase in neutrophil NO, a compound long known to stimulate guanylate cyclase," and which has recently been identified as the endothelial-derived relaxing factor or EDRF." Because the rise in NO is transient (due to GKINASE COMPARTMENTMUAWN IN NEUTROeHlLS -7. 617 8wk.oft)lmcLsM imogom (0through D) of nMl0philo pucncuatod with &Brcydk O W for 16 minuton and nimulotod for 2.6 minutoswith MLP. O-khu- io fodb k a C hod within tho nuckum and MTOC. D)t(rWW i n ” Contro.1 is ohown in (A) (origiMI nugnbtion x 2.100). the lability of the radical), it is likely that cyclic G M P increases arc also transient. Kaplan et at’ have shown that chemotaxis is inhibited by blocking NO synthesis with the competitive inhibitor of L-arginine. N”-monomethyl-Larginine. Inhibition of chemotaxis could be abrogated by L-arginine or dibutryl-cyclic GMP. These data support the involvement of cyclic G M P and G-kinase in regulating chemotaxis. Thus, the greater effectiveness of 8-Br-cyclic GMP in augmenting translocation may be due to its longer duration of action compared with cyclic GMP. Compartmentalitation of cyclic nucleotides in neutrophils is also supported by our observations that cyclic AMP and RI of A-kinase are compartmentalized within 30 seconds at the forming phagosome during phagocytosis? The concept of functional compartments in regulating cyclic nucleotide effectors has been described and/or postulated in other cellular systems.”u Further work is required to identify the targeting proteins for these effectors and determine the physiologic consequence. Immunocytochemistry. combined with biochemical analysis, offers an approach to correlate physiologic functions of intact cells with kinase modulation. A C K N M E D G M EN f We thank Dn M.Caplow. S.Earp. and K. Harden for review of the manuscript; Dr Bill Reed for his technical advice and revim of the manuscript: Dr Alton Steiner for amsultation; Dr Robert Bagncll for assistance with the confocal dual laser scanning miscope; and Elizabeth Parker for photographic assistance. Informed consent regarding the nature and possible consequences of the study was obtained from human subjects before donating blood. This investigation was approved by an Institutional Review Board and in accord with an assurance filed with. and approved by, the Department of Health and Human Services. REFERENCES 1. Estenm RD. Hill HR. Quie PG. Hogan N. Goldberg N D CyclicGMPand cell mavement. Nature 249458,1973 2. Simchovitt L, Fischbein LC. Spilberg 1. Atkinson J P Induction of a transient elevation in intracellular levels of adenosine-3’-5’cyclic monophosphate by chemotactic factors: An early event in human neutrophil activation. J lmmunol 124:1482.1980 3. Smolen JE. Korchak HM. 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