The vasodilator-stimulated phosphoprotein is regulated by cyclic GMP-dependent protein kinase during neutrophil spreading.

The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.

Chemotactic peptide-induced changes of intermediate filament organization in neutrophils during granule secretion: role of cyclic guanosine monophosphate.

In neutrophils activated to secrete with formyl-methionyl-leucyl-phenylalanine, intermediate filaments are phosphorylated transiently by cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). cGMP regulation of vimentin organization was investigated. During granule secretion, cGMP levels were elevated and intermediate filaments were transiently assembled at the pericortex to areas devoid of granules and microfilaments. Microtubule and microfilament inhibitors affected intermediate filament organization, granule secretion, and cGMP levels. Cytochalasin D and nocodazole caused intermediate filaments to assemble at the nucleus, rather than at the pericortex. cGMP levels were elevated in neutrophils by both inhibitors; however, with cytochalasin D, cGMP was elevated earlier and granule secretion was excessive. Nocodazole did not affect normal cGMP elevations, but specific granule secretion was delayed. LY83583, a guanylyl cyclase antagonist, inhibited granule secretion and intermediate filament organization, but not microtubule or microfilament organization. Intermediate filament assembly at the pericortex and secretion were partially restored by 8-bromo-cGMP in LY83583-treated neutrophils, suggesting that cGMP regulates these functions. G-kinase directly induced intermediate filament assembly in situ, and protein phosphatase 1 disassembled filaments. However, in intact cells stimulated with formyl-methionyl-leucyl-phenylalanine, intermediate filament assembly is focal and transient, suggesting that vimentin phosphorylation is compartmentalized. We propose that, in addition to changes in microfilament and microtubule organization, granule secretion is also accompanied by changes in intermediate filament organization, and that cGMP regulates vimentin filament organization via activation of G-kinase.

Compartmentalization of PDE-4 and cAMP-dependent protein kinase in neutrophils and macrophages during phagocytosis.

The compartmentalization of cAMP in human neutrophils during phagocytosis of serum-opsonized zymosan suggests that cAMP is an important second messenger for regulating phagocytosis. Type 4 cAMP-specific phosphodiesterase (PDE-4), cAMP-dependent protein kinase (PKA), and adenylate cyclase are the principal effector molecules for cAMP regulation in phagocytes. Immunofluorescence microscopy demonstrated that PDE-4 isoforms (HSPDE-4A, HSPDE-4B, HSPDE-4D) were targeted to the forming phagosome in neutrophils, and were colocalized with the catalytic subunit of PKA and degranulated myeloperoxidase. Phagocytosis and accumulation of PDE-4 and PKA near adherent zymosan were inhibited by elevating cAMP levels with forskolin or rolipram. cAMP, PDE-4, and PKA were localized at sites of zymosan adherence in cells treated with cytochalasin D to inhibit phagosome formation, suggesting that zymosan engagement to Fc/CR3 receptors triggers cAMP elevations at sites of phagocytosis. HSPDE-4A, HSPDE-4B, HSPDE-4D, and PKA also were localized at the forming phagosome in monocyte-derived macrophages, and the lysosomal marker CD63 demonstrated the absence of PDE-4 around internalized phagolysosomes. These results suggest that cAMP levels are focally regulated by PDE-4 at the nascent phagosome, and that PKA may phosphorylate proteins associated with pseudopodia formation and phagosome internalization.

Localization of cyclic GMP-dependent protein kinase in human mononuclear phagocytes.

The presence and physiological role of cGMP-dependent protein kinase (G-kinase) was investigated in human mononuclear phagocytes. Western blots of monocyte extracts revealed a single polypeptide band that comigrated with purified bovine lung G-kinase. G-kinase was localized by immunofluorescence microscopy in freshly isolated adherent human monocytes, monocyte-derived macrophages cultured from 4 to 14 days, and alveolar macrophages. In monocytes, G-kinase was localized in granules or vesicles in the cytoplasm, at the microtubule organizing center, on filaments, and in the nucleus. In monocyte-derived macrophages, intense staining for G-kinase was found in the vicinity of the Golgi, in vesicles throughout the cytoplasm, and diffusely in the nucleus. Dual-label confocal laser scanning microscopy demonstrated that G-kinase was colocalized with the endoplasmic reticulum. For comparison, G-kinase was localized in alveolar macrophages that were adhered from 3 to 30 min. In these cells, G-kinase was prominent within the organelle-rich area pericortical to the nucleus. However, a well-defined area of intense staining was also observed at the cell periphery at early time points during adherence and spreading. Rhodamine-labeled phalloidin showed that this peripheral area was rich in F-actin. Cytochalasin D, but not nocodazole, inhibited G-kinase targeting to the cell margin. Furthermore, the guanylate cyclase inhibitor LY83583 inhibited alveolar macrophage spreading and staining for G-kinase at the cell periphery. These data suggest that G-kinase may play an important role in cGMP-mediated regulation involved in protein processing and cell motility.

Control of glucan-induced systemic granulomatosis by cyclosporine A.

This study has shown that cyclosporine A (CyA), under certain conditions, is a powerful inhibitor of intravascular and extravascular monocyte/macrophage accumulation. Experiments were carried out in Lewis rats in which intravenous injection of particulate glucan calls forth a striking granulomatous response in lung, liver, and spleen and produces a marked stimulation of splenic erythro- and myelopoiesis. In agreement with the results of others, there was also a considerable elevation in monocyte/macrophage chemoattractant levels in the bronchoalveolar lavage fluid, which is held to be a key reaction in the pathogenesis of the histologic lesions. Treatment of the animals with subcutaneous injections of CyA prevented the rise in the chemoattractant activity and suppressed the granulomatous organ infiltration as well as the splenic hemopoiesis. The findings supply new insights into the activities of CyA and would support its clinical use in macrophage-dominated diseases.

Cyclic guanosine monophosphate-dependent protein kinase is targeted to intermediate filaments and phosphorylates vimentin in A23187-stimulated human neutrophils.

The effects of the calcium ionophore, A23187, on human neutrophil activation were studied in relation to the signaling mechanism of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton after stimulation with A23187. Over a period of 5 minutes, G-kinase was transiently colocalized with the intermediate filament protein, vimentin. At 3 minutes' stimulation with A23187, colocalization of G-kinase and vimentin was predominantly confined to filaments that extended into the uropod. The time of colocalization of G-kinase and vimentin was reduced in the A23187-stimulated cell from 3 minutes to 1 minute by 8-Br-cGMP. Coincident with colocalization was an increase in cGMP levels and transient phosphorylation of vimentin in adhered A23187-stimulated cells. Phosphorylation of vimentin was maximal after 3 minutes with A23187, and was essentially over at 5 minutes. The time of phosphorylation of vimentin was also reduced from 3 minutes to 1 minute when cells were preincubated with 8-Br-cGMP and then stimulated with A23187, which suggests that cyclic adenosine monophosphate (cAMP)-dependent protein kinase does not phosphorylate vimentin in A23187-treated neutrophils. Phosphorylation of vimentin was not observed in nonactivated cells treated only with 8-Br-cGMP. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation in A23187-treated cells, which provides supportive data that protein kinase C is not the phosphorylating enzyme. These results suggest that vimentin and G-kinase are colocalized in a Ca(2+)-dependent manner in neutrophils, and that vimentin is transiently phosphorylated by G-kinase in response to the colocalization of the two proteins. The transient redistribution of compartmentalized G-kinase represents one type of neutrophil activation mechanism.

High voltage immunoelectron microscopy of complement receptor type 3-mediated capping and internalization of group A streptococcal cell walls by human neutrophils.

The mechanism of human neutrophil clearance of peptidoglycan group A-specific polysaccharide polymers derived from streptococcal cell walls (PG-APS) was investigated by high voltage immunoelectron microscopy (HVEM) in order to determine how neutrophils process this highly inflammatory bacterial debris. Neutrophil monolayers were incubated from 5-30 min with serum-opsonized PG-APS. Cells were lightly fixed with 0.5% glutaraldehyde, and the PG-APS was localized on the neutrophil surface by immunogold using antibodies to N-acetyl-glucosamine and 15 nm colloidal gold coupled to goat anti-rabbit IgG. Neutrophils were viewed unsectioned by stereo HVEM. Patches of PG-APS were distributed randomly on the plasmalemma of well-spread neutrophils within 5 min. In polarized cells, PG-APS was densely localized on the uropod and retraction fibers. Within 15 min, PG-APS was predominantly concentrated into a large aggregate, measuring approximately 1 micron in diameter, near the cell margin or nucleus. The aggregate of PG-APS was engulfed in the vicinity of the indentation of the nucleus (hof). Intact microfilaments were required for aggregation and internalization of PG-APS. Binding of PG-APS was dependent upon complement fixation. Furthermore, PG-APS elicited an increase in density of complement receptor type 3 (CR3, C3bi receptor) on the neutrophil surface as determined by morphometry of immunogold labeled anti-CR3. When cells were stained for both PG-APS and CR3, co-localization was observed, and stereomicroscopy revealed clusters of CR3 in areas associated with phagocytosis. These data suggest that neutrophils use an efficient mechanism for removal of bacterial debris. Unlike whole streptococci which are phagocytosed at multiple sites, these bacterial cell walls are first collected into a large aggregate, or cap, which is then internalized at one site.

A comparative study of frozen-section immunoperoxidase and flow cytometry for immunophenotypic analysis of lymph node biopsies.

Immunophenotyping by flow cytometry and frozen-section immunoperoxidase was compared on 21 consecutive lymph node biopsy specimens, of which a diagnosis of lymphoma was made for 11 specimens. Samples for flow cytometry were obtained by a fine-needle aspiration technique. Concordance between frozen-section immunoperoxidase and flow cytometry for all routine markers on all specimens ranged from 76 to 100%. In general, B-cell markers showed poorer concordance than T-cell markers, with kappa and lambda light chains having the poorest concordance, at 76% each. Flow cytometry was significantly more sensitive (90 versus 30%; P < 0.006) and had a significantly higher negative predictive value (100 versus 63%; P < 0.006) than frozen-section immunoperoxidase for demonstrating light-chain restriction. There was no significant difference in the specificities (100 versus 91%) or positive predictive values (100% each) between the two methods. Both methods demonstrated characteristic immunophenotypes for intermediate cell lymphomas, small lymphocytic lymphomas, and T-cell lymphoblastic lymphomas. Frozen-section immunoperoxidase and flow cytometry appear to be significantly concordant methods for immunophenotypic analysis of lymph node biopsies. Light-chain restriction is more readily demonstrated by flow cytometry than frozen-section immunoperoxidase. We believe that ex vivo fine-needle aspiration is a simple and reliable method of obtaining cell suspensions of lymph nodes for flow cytometry.

Regulation of human neutrophil degranulation by LY-83583 and L-arginine: role of cGMP-dependent protein kinase.

The effects of guanosine 3',5'-cyclic monophosphate (cGMP) on the secretory response of activated human neutrophils were investigated using LY-83583, an inhibitor of soluble guanylate cyclase, and L-arginine, the precursor of nitric oxide formation. A 30% release of myeloperoxidase (MPO) and lactoferrin (LF) from the primary and specific granules, respectively, was detected by enzyme-linked immunosorbent assay in adhered neutrophils stimulated with 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 20 microM A-23187. LY-83583 (100 microM) inhibited the release of both LF and MPO after stimulation with FMLP or A-23187. Conversely, preincubation of neutrophils with 0.5 mM L-arginine augmented the release of LF and MPO in FMLP- and A-23187-stimulated cells. Concurrent with the increase in the degranulation response was an elevation of cGMP levels in L-arginine-treated cells, while stimulated cGMP levels were reduced in LY-83583-treated cells. Furthermore, cGMP-dependent protein kinase (G-kinase) activity was reduced in LY-83583-treated cells, as determined by the delay in G-kinase translocation to intermediate filaments and the inhibition of vimentin phosphorylation. Degranulation, elevation of cGMP levels, and targeting of G-kinase were also dependent on the concentration of A-23187 or FMLP. These data suggest that activators of neutrophil degranulation mediate this response through a cGMP-dependent protein kinase mechanism.

Suppression of local and systemic responses in streptococcal cell wall-induced acute inflammation of the air pouch by cyclosporine A. Comparison with the effects of two anti-inflammatory bis-benzimidazoles.

Injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide into a subcutaneous air pouch causes local outpouring of neutrophils and macrophages and distant hemopoietic proliferation in spleen and bone marrow. Cyclosporine A (CyA) suppressed neutrophil accumulation and all cell lines of hemopoiesis. trans-1,2-Bis(5-amidino-2-benzimidazolyl)ethene (BBE) also interfered with neutrophil exudation, yet reduced only the erythroid component of the hemopoietic process. The ethane analogue of BBE, on the other hand, did not prevent neutrophil emigration, but held down splenic erythropoiesis and myelopoiesis. All three compounds stimulated streptococcus group A cell wall-derived peptidoglycan polysaccharide uptake by pouch macrophages. CyA being the least active, BBE and its ethane analogue also produced a shift of wear-and-tear pigment from large numbers of small splenic macro-phages into small numbers of large macrophages. The pouch model is very useful in the study of anti-inflammatory compounds and has furnished the first evidence of CyA interference with massive neutrophilic infiltration and with hemopoietic signals.

Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells.

The role of cGMP-dependent protein kinase in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells was examined by studying the effects of cGMP on the phosphorylation of the Ca(2+)-ATPase regulatory protein phospholamban. Cultured rat aortic smooth muscle cells incubated with atrial natriuretic peptide II or sodium nitroprusside responded with increased phosphorylation of the 6000-Da subunit of phospholamban. The identity of phospholamban was confirmed using immunoprecipitation methods. Phosphorylation was associated with an increase in the activation of membrane-associated ATPase by Ca2+. These results indicated that at least one site of action of cGMP in smooth muscle cells is the sarcoplasmic reticulum, where phosphorylation of proteins regulating Ca2+ fluxes occurs. Studies using confocal laser scanning microscopy to define the cellular distribution of cGMP-dependent protein kinase suggested that the enzyme was localized to the same cellular region(s) as was phospholamban. Phosphorylation of proteins by cGMP in broken cell fractions from rabbit aorta was also performed. Phospholamban and other proteins were phosphorylated in the presence of cGMP but not cAMP, suggesting that only cGMP-dependent protein kinase was associated with smooth muscle membrane fractions containing phospholamban. These results suggest that one mechanism of action of cGMP in the reduction of intracellular Ca2+ is the activation of sarcoplasmic reticulum Ca(2+)-ATPase via phosphorylation of phospholamban. The data also support the concept that compartmentalization of protein kinases with substrates in the intact cell is an important factor involved in protein phosphorylation.

Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils.

The effects of cGMP-dependent protein kinase (G-kinase), a major cellular receptor of cGMP, were investigated in activated human neutrophils. Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton and nucleus after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), and transiently co-localized with the intermediate filament protein, vimentin. During this time period, the most remarkable co-localization of G-kinase and vimentin was observed between 1-2.5 min stimulation with fMLP. At that time co-localization of G-kinase and vimentin was predominantly confined to filaments which extended from regions adjacent to the nucleus into the uropod. Distinctive localization for only G-kinase was observed at the microtubule organizing center and euchromatin of the nucleus. The filamentous staining pattern for G-kinase and vimentin was enhanced in the presence of 8-Br-cGMP. Coincident with co-localization of G-kinase and vimentin in adherent neutrophils was a transient increase in cGMP levels and an increase in the phosphorylation of vimentin in fMLP-stimulated cells. The increase in cGMP levels was dependent upon cell adherence, was enhanced by preincubating neutrophils with L-arginine (the precursor for nitric oxide synthesis), and attenuated with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Phosphorylation of vimentin in the fMLP-stimulated neutrophil was observed in the presence or absence of exogenous cGMP, although in the presence of low concentrations of 8-Br-cGMP a more rapid phosphorylation of vimentin was observed that correlated with the enhanced co-localization of G-kinase and vimentin. Phosphorylation of vimentin was not observed in non-activated cells treated with 8-Br-cGMP, suggesting that phosphorylation only occurs when G-kinase is co-localized with vimentin. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation during fMLP stimulation, while 8-Br-cGMP enhanced phosphorylation in fMLP-treated cells. This suggests that neither protein kinase C nor cAMP-dependent protein kinase catalyze the phosphorylation of vimentin in neutrophils activated by fMLP. These results indicate that vimentin and G-kinase are co-localized in neutrophils and that vimentin is phosphorylated by G-kinase in response to the co-localization of the two proteins. A model for the targeting of G-kinase and vimentin is presented which hypothesizes that the transient redistribution of G-kinase may regulate neutrophil activation.

Streptococcal cell wall-induced systemic disease. Beneficial effects of trans-bis(5-amidino-2-benzimidazolyl)ethene, a novel, macrophage-directed anti-inflammatory agent.

Previously bis(5-amidino-2-benzimidazolyl)methane (BABIM) was identified as a strong inhibitor of the multisystem inflammatory disease induced in Lewis rats by injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide (PG-APS). A BABIM derivative, trans-bis(5-amidino-2-benzimidazolyl)ethene (BBE), has attracted attention because of striking qualitative and quantitative differences in its activities when compared with the parent compound. BBE could control destructive tibial osteitis and necrotizing granulomatous splenitis and hepatitis, regardless if given in a preventive or curative mode. The compound had little effect on synovitis, however. BABIM, on the other hand, was active against synovitis and osteitis, but not against splenic granuloma formation. To be effective, it needed to be applied in a preventive mode. BBE caused a characteristic enlargement of PG-APS-laden splenic and hepatic macrophages suggesting that those cells represent targets of the inhibitor. BBE may be a powerful tool for the study of granulomatous lesions.

KT5823 activates human neutrophils and fails to inhibit cGMP-dependent protein kinase phosphorylation of vimentin.

The effectiveness of the cGMP-dependent protein kinase inhibitor, KT5823, was investigated in human neutrophils. KT5823 did not inhibit the cGMP-dependent protein kinase mediated in vitro, or in situ phosphorylation of vimentin, a known substrate for this enzyme in activated neutrophils. In addition, KT5823 was shown to induce dramatic shape changes in neutrophils, suggesting it has an activating effect upon the cells.

Application of confocal scanning laser microscopy in experimental pathology.

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out-of-focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM-imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein-A is the major non-serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein-A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid-phase pinocytosis. The blood-nerve barrier (BNB) in peripheral nerves may be an important target during toxin-induced neuropathies. Ricin-induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)-dextran into the endoneurial compartment.

Phorbol ester induces transient focal concentrations of functional, newly expressed CR3 in neutrophils at sites of specific granule exocytosis.

Treatment of neutrophils with phorbol myristate acetate (PMA) increases surface expression of CR3 (iC3b-receptor; CD11b/CD18). This up-regulation was examined in whole-mount preparations of adherent neutrophils by stereo high voltage immunoelectron microscopy. In the absence of PMA, immunogold-labeled CR3 was uniformly distributed in the plasma membrane. After 5 to 15-min incubations with PMA, when the highest rates of specific granule exocytosis occurred, the average density of CR3 in the membrane doubled. During this time, dense foci of CR3 were observed in addition to the uniform distribution of CR3. These dense concentrations of CR3 colocalized with secreted lactoferrin (LF), a specific granule marker, above assemblies of cytoplasmic vesicles that were morphologically similar to specific granules and contained LF. After longer incubations in PMA, LF secretion ceased, LF staining became rare, and the high density areas of CR3 were no longer present. These data demonstrate that incipient CR3 appear on the cell surface in high concentration at sites of specific granule exocytosis and then diffuse out into the plasmalemma. PMA also induced shedding of CR3 from the cell surface at the cell margins on structures which also contained LF. Shed CR3 was immunoprecipitated from incubation supernatants and shown to be of identical subunit composition to surface CR3. Although others have shown that the mobile, incipient surface CR3 do not mediate neutrophil adherence to endothelium or homotypic aggregation, the current experiments demonstrated that such CR3 do mediate iC3b-dependent adhesion. The rapid appearance and subsequent dissipation of high concentrations of CR3 on the neutrophil surface caused by specific granule fusion may be essential for neutrophil function at sites of complement deposition.

Compartmentalization of cyclic GMP-dependent protein kinase in formyl-peptide stimulated neutrophils.

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. Immunofluorescence microscopy 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 localization was investigated in neutrophils activated with N-formyl-methionyl-leucyl-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 polarized cells (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.

Sequential events in the pathogenesis of streptococcal cell wall-induced arthritis and their modulation by bis(5-amidino-2-benzimidazolyl)methane (BABIM).

This report builds on the authors' earlier discovery of bis(5-amidino-2-benzimidazolyl)methane (BABIM) as a strong suppressive agent for streptococcal cell wall fragment-induced arthritis in the Lewis rat. As a synthetic inhibitor of trypsinlike proteases, BABIM opens up a new route to the control of inflammatory joint disease. To gain a deeper insight into the function of the compound, the authors have now studied its influence on the sequential development of the joint changes and the associated lesions in spleen and liver. Bis(5-amidino-2-benzimidazolyl)methane is shown to block acute synovitis, to retard and reduce granuloma formation in spleen and liver, to decrease neutrophilic leukocytosis, and to diminish hemopoietic hyperplasia in the bone, and thus also to mitigate the distinctive osteoclastic and chondroclastic events. The compound does not interfere with the splenic immune response, the temporary rise in hepatocytic mitotic activity, or the organ deposition of streptococcal cell walls.