Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-alpha (TNFalpha). Although TNFalpha has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNFalpha is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNFalpha on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNFalpha was found to result in beta2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to beta2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNFalpha led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNFalpha-induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to beta2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNFalpha leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.     

Differential modulation of annexin I binding sites on monocytes and neutrophils

Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN). These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.     

Glycogen Synthase Kinase-3 and Omi/HtrA2 Induce Annexin A2 Cleavage followed by Cell Cycle Inhibition and Apoptosis

Annexin A2 is involved in multiple cellular processes, including cell survival, growth, division, and differentiation. A lack of annexin A2 makes cells more sensitive to apoptotic stimuli. Here, we demonstrate a potential mechanism for apoptotic stimuli-induced annexin A2 cleavage, which contributes to cell cycle inhibition and apoptosis. Annexin A2 was persistently expressed around the proliferative but not the necrotic region in BALB/c nude mice with human lung epithelial carcinoma cell A549-derived tumors. Knockdown expression of annexin A2 made cells susceptible to either serum withdrawal-induced cell cycle inhibition or cisplatin-induced apoptosis. Under apoptotic stimuli, annexin A2 was time-dependently cleaved. Mechanistic studies have shown that protein phosphatase 2A (PP2A)-activated glycogen synthase kinase (GSK)-3 is essential for this process. Therefore, inhibiting GSK-3 reversed serum withdrawal-induced cell cycle inhibition and cisplatin-induced apoptosis. Furthermore, inhibiting serine proteases blocked apoptotic stimuli-induced annexin A2 cleavage. Bax activation and Mcl-1 destabilization, which is regulated by PP2A and GSK-3, caused annexin A2 cleavage via an Omi/HtrA2-dependent pathway. Taking these results together, we conclude that GSK-3 and Omi/HtrA2 synergistically cause annexin A2 cleavage and then cell cycle inhibition or apoptosis.     

Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.     

Divergent effects of alpha1-antitrypsin on neutrophil activation, in vitro

alpha1-Antitrypsin (AAT) is a major circulating serine proteinase inhibitor in humans. The anti-proteinase activity of AAT is inhibited by chemical modification. These include inter- or intramolecular polymerisation, oxidation, complex formation with target proteinases (e.g., neutrophil elastase), and/or cleavage by multi-specific proteinases. In vivo, several modified forms of AAT have been identified which stimulate biological activity in vitro unrelated to inhibition of serine proteinases. In this study we have examined the effects of native and polymerised AAT and C-36 peptide, a proteolytic cleavage product of AAT, on human neutrophil activation, in vitro. We show that the C-36 peptide displays striking concentration-dependent pro-inflammatory effects on human neutrophils, including induction of neutrophil chemotaxis, adhesion, degranulation, and superoxide generation. In contrast to C-36 peptide, native and polymerised AAT at similar and higher concentrations showed no effects on neutrophil activation. These results suggest that cleavage of AAT may not only abolish its proteinase inhibitor activity, but can also generate a powerful pro-inflammatory activator for human neutrophils.     

Adiponectin inhibits neutrophil apoptosis via activation of AMP kinase, PKB and ERK 1/2 MAP kinase

Neutrophils are abundant, short-lived leukocytes that play a key role in the immune defense against microbial infections. These cells die by apoptosis following activation and uptake of microbes and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Adiponectin exerts anti-inflammatory effects on neutrophil antimicrobial functions, but whether this abundant adipokine influences neutrophil apoptosis is unknown. Here we report that adiponectin in the physiological range (1–10 μg/ml) reduced apoptosis in resting neutrophils, decreasing caspase-3 cleavage and maintaining Mcl-1 expression by stabilizing this anti-apoptotic protein. We show that adiponectin induced phosphorylation of AMP-activated kinase (AMPK), protein kinase B (PKB), extracellular signal-regulated kinase (ERK 1/2) and p38 mitogen activated protein kinase (MAPK). Pharmacological inhibition of AMPK, PKB and ERK 1/2 ablated the pro-survival effects of adiponectin and treatment of neutrophils with an AMPK specific activator (AICAR) and AMPK inhibitor (compound C) respectively decreased and increased apoptosis. Finally, activation of AMPK by AICAR or adiponectin also decreased ceramide accumulation in the neutrophil cell membrane, a process involved in the early stages of spontaneous apoptosis, giving another possible mechanism downstream of AMPK activation for the inhibition of neutrophil apoptosis.     

Luteolin inhibits pyrogallol-induced apoptosis through the extracellular signal-regulated kinase signaling pathway

Luteolin is an antioxidative, antitumor and anti-inflammatory flavone. It has been shown to reduce endothelial dysfunction, but the mechanism is not clear. We set out to explore the effects of luteolin on apoptosis and its mechanism of action in endothelial cells. The effect of luteolin on pyrogallol-induced superoxide stress and the subsequent apoptosis was studied in the mouse heart capillary endothelial cell line H5V and human umbilical vein endothelial cells, by the use of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Hoechst staining, and western blot. Pyrogallol (0-400 μm) dose-dependently induced reactive oxygen species production, cytotoxicity, an annexin V-fluorescein isothiocyanate increase, mitochondrial transmembrane depolarization and DNA condensation in both H5V and human umbilical vein endothelial cells; these actions were reversed by luteolin (0.78-50 μm) in a concentration-dependent manner. Luteolin suppressed the poly (ADP-ribose) polymerase activation, caspase-8 cleavage and p38 mitogen-activated protein kinase activation triggered by pyrogallol, and stimulated the extracellular signal-regulated kinase signaling pathway to counteract the pyrogallol-induced apoptotic signals. Luteolin is an effective agent for the protection of endothelial cells from superoxide stress-induced apoptosis via the extracellular signal-regulated kinase signaling pathway.     

Quantification of annexin I in subcellular fractions of human neutrophils reveals an exclusive cytosolic localisation

Annexin I is an abundant cytosolic protein in human neutrophils. Besides its intracellular location, annexin I is found as an extracellular protein and the pathway for secretion has been of interest since the protein lacks a signal sequence for secretion. It was recently shown that annexin I is stored in the secretory gelatinase granules of human neutrophils, suggesting that the protein might be released through a granule mobilisation and fusion process resembling classical secretion. In this study we have determined the intracellular localisation of annexin I in human neutrophils using subcellular fractionation, protein separation by SDS-PAGE and immunoblotting, and show that virtually all annexin I is localised in the cell cytosol.     

Endogenous cleavage of annexin I generates a truncated protein with a reduced calcium requirement for binding to neutrophil secretory vesicles and plasma membrane

We have earlier shown that an N-terminal truncated annexin I molecule, annexin I(des1-8), is generated in human neutrophils through cleavage by a membrane localized metalloprotease. The truncated protein showed differences in membrane binding among the neutrophil granule populations as compared to full-length annexin I. In this study, we investigated the cleavage capabilities of isolated neutrophil secretory vesicles and plasma membrane, and the binding of full-length annexin I and annexin I(des1-8) to these membrane fractions. Translocations were performed in vitro to secretory vesicles and plasma membrane, respectively, at different Ca(2+) concentrations. We show that the annexin I-cleaving membrane localized metalloprotease is present both in the secretory vesicles and the plasma membrane. The N-terminal truncation of annexin I gives rise to a molecule with a decreased Ca(2+) requirement for binding, both to secretory vesicles and plasma membrane. There was, thus, no difference in binding of either full-length annexin I or annexin I(des1-8) to the secretory vesicles as compared to the plasma membrane.     

Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products

The coronavirus Avian infectious bronchitis virus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly(673)-Gly(674) dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly(2265)-Gly(2266) dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn(2313) residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q(2583)-G(2584)) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product.     

Annexin A1 on the Surface of Early Apoptotic Cells Suppresses CD8+ T Cell Immunity

Prevention of an immune response against self-antigens derived from apoptotic cells is essential to preclude autoimmune and chronic inflammatory diseases. Here, we describe apoptosis induced externalization of endogenous cytosolic annexin 1 initiating an anti-inflammatory effector mechanism that suppresses the immune response against antigens of apoptotic cells. Cytosolic annexin 1 rapidly translocated to the apoptotic cell surface and inhibited dendritic cell (DC) activation induced by Toll like receptors (TLR). Annexin 1-inhibited DC showed strongly reduced secretion of pro-inflammatory cytokines (e.g. TNF and IL-12) and costimulatory surface molecules (e.g. CD40 and CD86), while anti-inflammatory mediators like PD-L1 remained unchanged. T cells stimulated by such DC lacked secretion of interferon-γ (IFN-γ) and TNF but retained IL-10 secretion. In mice, annexin 1 prevented the development of inflammatory DC and suppressed the cellular immune response against the model antigen ovalbumin (OVA) expressed in apoptotic cells. Furthermore, annexin 1 on apoptotic cells compromised OVA-specific tumor vaccination and impaired rejection of an OVA-expressing tumor. Thus, our results provide a molecular mechanism for the suppressive activity of apoptotic cells on the immune response towards apoptotic cell-derived self-antigens. This process may play an important role in prevention of autoimmune diseases and of the immune response against cancer.     

The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid

The Ca2+/phospholipid-dependent protein kinase (protein kinase C) of human neutrophils is converted to a proteolytically modified Ca2+/phospholipid-independent form (Inoue, M., Kishimoto, A., Takai, Y.U., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) on incubation with neutrophil membranes in the presence of micromolar concentrations of Ca2+ and an endogenous Ca2+-requiring proteinase (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6435-6439). We have now demonstrated the appearance of a similar Ca2+/phospholipid-independent kinase in intact human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA). The following evidence supports the conclusion that the Ca2+/phospholipid-independent protein kinase recovered from the PMA-treated cells is a proteolytically modified form of the "native" protein kinase C. 1) In cells exposed to PMA, the rate of disappearance of Ca2+/phospholipid-dependent protein kinase C activity is correlated with the rate of appearance of the Ca2+/phospholipid-independent kinase. 2) The chromatographic behavior of the new protein kinase and its molecular size (approximately 65 kDa) are identical to those previously reported for the proteolytically modified form of protein kinase C. 3) The modified protein kinase no longer binds to the cell membrane and is recovered almost entirely in the cytosol fraction. 4) In neutrophils preloaded with inhibitors of the Ca2+-requiring proteinase, stimulation with PMA results in translocation of protein kinase C from the cytosol fraction to the particulate fraction, but the appearance of the soluble, Ca2+/phospholipid-dependent form is prevented. We conclude that binding of protein kinase C to the plasma membrane and its proteolytic conversion are related, but independent, processes both elicited by exposure of neutrophils to the phorbol ester. Proteolytic cleavage of the membrane-bound protein kinase C provides an alternative mechanism for its activation and may account for certain of the cellular responses observed in PMA-stimulated neutrophils.     

Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal

Annexin A1 is an intracellular calcium/phospholipid-binding protein that is involved in membrane organization and the regulation of the immune system. It has been attributed an anti-inflammatory role at various control levels, and recently we could show that annexin A1 externalization during secondary necrosis provides an important fail-safe mechanism counteracting inflammatory responses when the timely clearance of apoptotic cells has failed. As such, annexin A1 promotes the engulfment of dying cells and dampens the postphagocytic production of proinflammatory cytokines. In our current follow-up study, we report that exposure of annexin A1 during secondary necrosis coincided with proteolytic processing within its unique N-terminal domain by ADAM10. Most importantly, we demonstrate that the released peptide and culture supernatants of secondary necrotic, annexin A1-externalizing cells induced chemoattraction of monocytes, which was clearly reduced in annexin A1- or ADAM10-knockdown cells. Thus, altogether our findings indicate that annexin A1 externalization and its proteolytic processing into a chemotactic peptide represent final events during apoptosis, which after the transition to secondary necrosis contribute to the recruitment of monocytes and the prevention of inflammation.     

Mapping of the Feline Calicivirus Proteinase Responsible for Autocatalytic Processing of the Nonstructural Polyprotein and Identification of a Stable Proteinase-Polymerase Precursor Protein

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.     

A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils

Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.     

Annexin I is stored within gelatinase granules of human neutrophil and mobilized on the cell surface upon adhesion but not phagocytosis

Annexin I, a member of the calcium- and phospholipid-binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co-localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.     

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.     

Modulation of phagocytosis of apoptotic neutrophils by supernatant from dexamethasone-treated macrophages and annexin-derived peptide Ac(2-26)

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac(2-26)) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mphi). Peptide Ac(2-26)-stimulated phagocytosis is accompanied by rearrangement of the Mphi actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mphi were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mphi significantly enhanced the ability of naive Mphi to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mphi from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.     

Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis

The recent appreciation of the role played by endogenous counterregulatory mechanisms in controlling the outcome of the host inflammatory response requires specific analysis of their spatial and temporal profiles. In this study, we have focused on the glucocorticoid-regulated anti-inflammatory mediator annexin 1. Induction of peritonitis in wild-type mice rapidly (4 h) produced the expected signs of inflammation, including marked activation of resident cells (e.g., mast cells), migration of blood-borne leukocytes, mirrored by blood neutrophilia. These changes subsided after 48-96 h. In annexin 1(null) mice, the peritonitis response was exaggerated ( approximately 40% at 4 h), with increased granulocyte migration and cytokine production. In blood leukocytes, annexin 1 gene expression was activated at 4, but not 24, h postzymosan, whereas protein levels were increased at both time points. Locally, endothelial and mast cell annexin 1 gene expression was not detectable in basal conditions, whereas it was switched on during the inflammatory response. The significance of annexin 1 system plasticity in the anti-inflammatory properties of dexamethasone was assessed. Clear induction of annexin 1 gene in response to dexamethasone treatment was evident in the circulating and migrated leukocytes, and in connective tissue mast cells; this was associated with the steroid failure to inhibit leukocyte trafficking, cytokine synthesis, and mast cell degranulation in the annexin 1(null) mouse. In conclusion, understanding how inflammation is brought under control will help clarify the complex interplay between pro- and anti-inflammatory pathways operating during the host response to injury and infection.     

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.