Dual action of cAMP-dependent protein kinase on granulocyte movement.

Cell locomotion is a continuous cycle of integrin-dependent attachments and detachments along chemotactic gradients, driven by dynamic modulations of the actin network. Cyclic AMP (cAMP), which is known to be generated by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) receptors but not by beta2 integrins, was investigated as a coordinator of granulocyte locomotion. Elevation of cAMP by exposure to forskolin (100 microM) and 1-isobutyl-methylxanthine (IBMX; 100 microM) caused a marked reduction in beta2 integrin-induced polymerisation of actin, but had a less pronounced effect on the fMLP-induced actin response. Pretreatment of cells with rp-adenosine-3',5'-cyclic monophosphorothioate (rp-cAMPS; 50 microM), an inhibitor of the cAMP-dependent protein kinase (cAPK), resulted in a significant increase in the fMLP-induced actin polymerisation response. In agreement with the effect on filamentous actin (F-actin) forskolin and IBMX markedly suppressed the migration of granulocytes towards fMLP. Surprisingly enough, pretreatment of cells with rp-cAMPS inhibited cell movement to the same extent as forskolin and IBMX did. This dual action of cAMP on granulocyte migration suggest an important regulatory mechanism whereby the balance of this intracellular signal results in an optimal locomotory response.

Engagement of L-selectin impairs the actin polymerizing capacity of beta 2-integrins on neutrophils.

A sequential activation of L-selectin and beta 2-integrins on neutrophils is crucial for the rolling, adherence and subsequent migration of these cells on the endothelium. However, little is known about a possible interplay between these adhesion receptors in the final regulation of cell motility. The results presented here show that sulfatides themselves (here used as tools to activate L-selectins), have no major effect on the cellular content of filamentous actin (F-actin), but cause a time-related decrease in the beta 2-integrin-induced formation of F-actin. This effect of sulfatides was abolished in cells lacking L-selectin as a result of pretreatment with chymotrypsin. A similar sulfatide-induced activation of L-selectin also caused a pronounced and time-related decrease of a subsequent chemotactic peptide-induced F-actin response. The effect of sulfatides on both beta 2-integrin- and chemotactic peptide-induced F-actin were abolished if L-selectin were blocked by preincubating the cells with specific antibodies to L-selectin. These effects of L-selectin engagement on cellular F-actin content were neither abolished by blocking the cytosolic free Ca2+ signal with bis-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraaceticacid tetraacetoxymethyly ester (MAPT/AM) nor by blocking a cAMP-induced activation of protein kinase A by pretreating the cells with adenosine-3',5'-cyclic monophos-phorothioate (Rp-cAMPS). Instead we found that L-selectin engagement impaired an early beta 2-integrin-induced tyrosine kinase activation, an event shown to be necessary for a normal beta 2-integrin-mediated F-actin response. The present demonstration of a negative feed-back function of L-selectin on beta 2-integrin-induced modulations of the actin cytoskeleton, suggests that the relative distribution and/or density of the respective L-selectin and beta 2-integrin ligands on endothelial cells might be important factors in determining the final site of firm adhesion and extravasation of neutrophils.

Inhibitors of farnesyl and geranylgeranyl methyltransferases prevent beta 2 integrin-induced actin polymerization without affecting beta 2 integrin-induced Ca2+ signaling in neutrophils.

The role of prenylated proteins such as low molecular weight G-proteins (LMW G-proteins) in beta 2 integrin-dependent neutrophil signal transduction was investigated using two methyltransferase inhibitors, N-Acetyl-S-farnesyl-L-cysteine (AFC) and N-acetyl-s-geranylgeranyl-L-cysteine (AGGC), and an inactive control, N-acetyl-S-geranyl-L-cysteine (AGC). The drugs did not affect beta 2 integrin-induced protein tyrosine phosphorylations or cytosolic calcium transients. However, AGGC inhibited beta 2 integrin-induced actin polymerization (IC50 of approximately 45nM), as did AFC(IC50 of approximately 5.5 microM), but not AGC. Thus, prenylated proteins, such as LMW G-proteins, are responsible for beta 2 integrin regulation of actin filament reorganization downstream of tyrosine kinase(s) activation, and represent a beta 2 integrin signaling mechanism distinct from the pathway which regulates cytosolic calcium transients.

Beta 2 integrin engagement triggers actin polymerization and phosphatidylinositol trisphosphate formation in non-adherent human neutrophils.

Beta 2 integrins are involved in the adhesion of leukocytes to other cells and surfaces. Although adhesion is required for cell locomotion, little is known regarding the way beta 2 integrin-receptors affect the actin network in leukocytes. In the present study filamentous actin (F-actin) levels in non-adherent human neutrophils have been measured by phalloidin staining after antibody cross-linking of beta 2 integrins. Antibody engagement of beta 2 integrins resulted in a rapid and sustained (146 and 131% after 30 and 300 s, respectively) increase in the neutrophil F-actin content. This is in contrast to stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), which causes a prompt and pronounced but rapidly declining rise in F-actin (214 and 127% after 15 and 300 s, respectively). Priming neutrophils with 1 nM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the beta 2 integrin-induced response but had no effect on the kinetics (199% after 30 s and 169% after 300 s). Removal of extracellular Ca2+ only marginally affected the beta 2 integrin-induced F-actin response for cells that were pretreated with PMA whereas the response for nonprimed cells was reduced by half. This suggests that even though extracellular Ca2+ has a modulatory effect it is not an absolute requirement for beta 2 integrin-induced actin polymerization. beta 2 integrin engagement did not affect the resting cellular level of cAMP arguing against a role of cAMP in beta 2 integrin-induced actin assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium signaling capacity of the CD11b/CD18 integrin on human neutrophils.

The CD11b/CD18 integrin is a major cell adhesion molecule of myelomonocytic cells. Exposure of human neutrophils in suspension to CD11b or CD18 monoclonal antibodies (mAbs)2 does not affect the resting level of cytosolic free Ca2+ in these cells; however, a subsequent cross-linking of either of these antibodies triggers a prompt and significant cytosolic-free Ca2+ transient lasting about 10 min. The rise in cytosolic-free Ca2+ (from 130 +/- 2 to 414 +/- 12 nM or 111 +/- 12 to 331 +/- 22 nM caused by cross-linking of CD11b or CD18 subunits, respectively) is due to both mobilization of Ca2+ from intracellular stores and influx of Ca2+ across the plasma membrane. Cross-linking of the common leukocyte antigen (CD45) did not alter the basal level of cytosolic free Ca2+. In accordance with other adherence-induced phenomena and with CD11/CD18-mediated phagocytosis, these Ca2+ signals were only modestly affected by pertussis toxin. Thus, the present data clearly indicate that the CD11b/CD18 integrin on human neutrophils is capable of inducing a prompt cytosolic-free Ca2+ signal. These findings directly support the recent suggestion that the CD11b/CD18 integrin is responsible for the "spontaneous oscillations" of cytosolic-free Ca2+ observed in adherent neutrophils and, at least partially, also explain how integrin-mediated adherence can modify the functional responsiveness of neutrophils to a subsequent agonist stimulation.

Tumor necrosis factor-induced degranulation in adherent human neutrophils is dependent on CD11b/CD18-integrin-triggered oscillations of cytosolic free Ca2+.

We have recently been able to correlate closely the "spontaneous" oscillatory activity of cytosolic free Ca2+ in adherent human neutrophils with the ability of tumor necrosis factor (TNF) to induce secretion of granule proteins from these cells. In the present work we show with a single-cell technique that preincubation of human neutrophils with antibodies to CD18, the common beta chain of leukocyte adhesion proteins, inhibits TNF-induced secretion of lactoferrin in a time- and concentration-dependent manner. Similar effects of CD18 antibodies were found on chemotactic factor (fMet-Leu-Phe)- but not on phorbol 12-myristate 13-acetate-induced secretion, suggesting that cell-surface-receptor-mediated secretion is dependent on integrin-associated signals. Similarly, antibodies to CD11b (alpha chain of macrophage 1) also inhibited TNF- and fMet-Leu-Phe- but not phorbol 12-myristate 13-acetate-stimulated release of lactoferrin. Antibodies to CD11a (alpha chain of lymphocyte function-associated antigen 1) or CD11c (alpha chain of p150,95) had only a minimal effect on agonist-induced secretion. Data obtained in several laboratories, including our own, made us suspect that integrin interaction with the surface is responsible for the oscillatory activity of cytosolic free Ca2+ in adherent cells. Indeed, preincubation with antibodies to either CD18 or CD11b, but not to CD11c, inhibited the oscillations of cytosolic free Ca2+ in adherent neutrophils. This inhibitory effect was evident both as a reduction of the number of responding cells and as a reduction of the oscillatory activity in the cells. In conclusion, the oscillatory activity of cytosolic free Ca2+ in adherent neutrophils is mediated through the CD18/CD11b integrins. The generation of this Ca2+ signal may explain how adherence, by way of the integrins, changes the functional properties of the cell and enables TNF to induce secretion.