Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.     

Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin

Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that alpha5beta1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil beta1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the beta1 integrins. Antibodies or peptides that block alpha5beta1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block beta1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated beta1 integrins.     

Beta2 integrin-dependent phosphorylation of protein-tyrosine kinase Pyk2 stimulated by tumor necrosis factor alpha and fMLP in human neutrophils adherent to fibrinogen.

Tumor necrosis factor alpha and fMLP can activate a broad range of cellular functions in neutrophils adherent to biological surfaces. These functions are mediated by integrins and involve the activation of tyrosine kinases. Here, we report that Pyk2, a member of the focal adhesion kinase family, was present in human neutrophils and was rapidly phosphorylated and activated following tumor necrosis factor alpha and fMLP stimulation in an adhesion-dependent manner. Tyrosine phosphorylation of Pyk2 was attenuated by beta2 integrin blocking with specific antibodies. The tyrosine phosphorylation of Pyk2 was downstream of protein kinases Lyn, Syk and protein kinase C and cytoskeletal organization. The activation of Pyk2 may play a role in adhesion/cytoskeleton-associated neutrophils function.     

Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.     

Mycobacterium lepraemurium activates macrophages but fails to trigger release of superoxide anion

Mycobacterium lepraemurium failed to stimulate a normal respiratory burst when presented to mouse peritoneal or bone marrow macrophages. By comparison, Mycobacterium bovis (strain Bacillus Calmette-Guerin) or Saccharomyces cerevisiae, as expected, stimulated macrophages to release a large amount of superoxide anion (O2-). M. lepraemurium did not interfere with the response to yeast when both microbes were added together to macrophages. The low release of O2- induced by M. lepraemurium was not due to failure of M. lepraemurium to activate or prime macrophages, because exposure of macrophages to M. lepraemurium caused the expected enhancement of O2- release when the macrophages were stimulated by PMA. Similarly, macrophages taken from mice infected with M. lepraemurium were activated, as indicated by high PMA-stimulated O2- release. Macrophages primed in vitro by exposure to Escherichia coli LPS for 24 h did show a moderate O2- response when stimulated by M. lepraemurium, but macrophages primed by exposure to IFN-gamma muramyl dipeptide, or M. lepraemurium showed a weak response when subsequently challenged with M. lepraemurium. The priming effect of M. lepraemurium or LPS decreased substantially after macrophages were cultured in fresh medium for 24 h. Heat killing or opsonization of M. lepraemurium caused the M. lepraemurium to stimulate a high amount of O2- release from LPS-primed macrophages, but heat killing or opsonization of M. lepraemurium had no effect on release of O2- from unprimed macrophages. The results suggest that M. lepraemurium is taken into macrophages by a mechanism that bypasses the FcR and other receptors that are capable of triggering the production of O2-.     

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)     

Resting murine neutrophils express functional alpha 4 integrins that signal through Src family kinases

There is mounting evidence that alpha(4) (CD49d) integrins are involved in neutrophil recruitment and function during inflammatory responses. We report that all resting murine neutrophils derived from bone marrow or peripheral blood express easily detectable levels of alpha(4) integrins on their surface. These alpha(4) integrins were functional, as demonstrated by stimulation of respiratory burst when neutrophils adhered to surfaces coated with the murine vascular cell adhesion molecule-1 (mVCAM-1). Adhesion occurred via alpha(4) integrins, as preincubation of neutrophils with an anti-alpha(4)-specific Ab inhibited attachment to mVCAM-1. Direct cross-linking of the alpha(4) integrin subunit by surface-bound mAbs also elicited superoxide release and release of the secondary granule marker, lactoferrin. The functional responses that occurred downstream of alpha(4) integrin cross-linking required signaling by Src family kinases. Neutrophils derived from hck(-/-)fgr(-/-)lyn(-/-) triple-knockout or hck(-/-)fgr(-/-) double-knockout mice failed to undergo respiratory burst when plated on mVCAM-1. Triple mutant neutrophils were also defective in release of both superoxide and lactoferrin when plated on surfaces coated with mAbs directed against alpha(4). Correlated with impaired alpha(4)-induced functional responses, triple-mutant neutrophils also failed to spread and tightly adhere to anti-alpha(4) mAb-coated surfaces. This is the first direct evidence that functional alpha(4) integrins are expressed by murine PMNs, and that these surface molecules can mediate cellular responses such as tight adhesion, spreading, sustained respiratory burst, and specific granule release in vitro. Moreover the alpha(4) integrins, like all other integrins tested, use the Src family kinases to transduce intracellular signals.     

Roles of beta 2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury.

The roles of beta 2 integrin molecules in neutrophil accumulation and tissue injury have been examined by the use of antibodies that are reactive with human CD11b and CD18 and cross-react with the homologous epitopes on rat neutrophils. Adherence to rat pulmonary artery endothelial cells by human neutrophils and endothelial cell killing by phorbol ester-activated human neutrophils required CD11b, CD11c, and CD18. Companion adherence studies between rat neutrophils and endothelial cells revealed a requirement for both CD11b and CD18. Neither anti-CD11b nor anti-CD18 depressed in vitro responses (O2- generation and chemotactic migration) of rat neutrophils. The accumulation of neutrophils in glycogen-induced peritoneal exudates was diminished substantially in rats treated with either anti-CD18 or anti-CD11b. In oxidant-mediated acute lung injury induced by rapid intravascular infusion of cobra venom factor, treatment of rats with either anti-CD18 or anti-CD11b significantly attenuated injury as assessed by increases in vascular permeability and hemorrhage. These protective effects correlated morphologically with diminished adhesion of neutrophils to interstitial intrapulmonary capillary endothelial cells. In studies of immune complex (BSA-anti-BSA)-induced alveolitis and dermal vasculitis, anti-CD18 had protective effects at all doses of anti-BSA employed. The protective effects of anti-CD18 correlated with diminished neutrophil accumulation in tissues at lower doses of anti-BSA. Although anti-CD11b was not effective under the same experimental conditions, intratracheal administration of this antibody conveyed protection against immune complex-induced lung injury, suggesting that both CD11b and CD18 are required for the full expression of injury. The current studies also demonstrated that when surface-bound IgG immune complexes were treated with fresh rat serum, the increment in O2- and TNF alpha generated by alveolar macrophages was suppressed by anti-CD18, but not by anti-CD11b, suggesting a heretofore unrecognized role for CD18 in the O2- and TNF-alpha responses of alveolar macrophages. Thus, neutrophil beta 2 integrins play a requisite role for the full expression of complement-dependent and oxygen radical-mediated injury of the lung and dermal vasculature.     

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.     

The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion.

Activated monocytic cells and neutrophils adhere to substrates coated with a wide variety of proteins including albumins, catalase, casein, and various extracellular matrix proteins. This adhesion can be specifically inhibited by antibodies directed to the beta 2 integrin subunit. This adhesion to protein substrates shares some similarities with two known protein-protein recognition systems with little apparent binding specificity, namely, the interactions of heat shock proteins and histocompatibility antigens with denatured proteins or peptides. Cell adhesion and affinity chromatography experiments were performed to test the hypothesis that monocytes and neutrophils adhere to and migrate on protein substrates due to the presence of cell surface receptors that recognize common protein structures such as denatured protein epitopes. Adhesion experiments revealed that activated monocytic cells adhere more rapidly and extensively on substrates coated with denatured protein versus native protein. Both adhesion and migration on such substrates in vitro was dependent on beta 2 integrins since blocking antibodies completely interfered with these cellular responses. Affinity chromatography experiments revealed that the Mac-1 and p150,95 integrins could be isolated from monocyte-differentiated HL-60 cells or neutrophils on a denatured protein-Sepharose column. Much greater yields of the receptors were obtained on a denatured versus native protein Sepharose column. The binding of these receptors was specific in that the LFA-1 beta 2 integrin did not bind to the denatured protein column. These data provide evidence that the adhesion of activated monocytes and neutrophils to many protein substrates in vitro is due to the ability of Mac-1 and p150,95 to directly bind to denatured proteins. A model of leukocyte adhesion and invasion whereby activated leukocytes denature extracellular proteins during diapedesis, making them suitable for recognition by beta 2 integrins, is proposed.     

Promotion of neutrophil adherence to human osteoblasts by microcrystals and f-Met-Leu-Phe

Human osteoblast-like cells (hOB) stimulated by monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) microcrystals produce the neutrophil chemoattractant IL-8. We investigated whether human neutrophils can adhere to hOB and respond to hOB preactivated by MSUM, CPPD, or by f-Met-Leu-Phe (fMLP). Confluent hOB were coincubated with human blood neutrophils in the presence of MSUM, CPPD or fMLP. MSUM, CPPD, and fMLP stimulated a significant adherence of neutrophils to hOB after a 1h incubation. This effect was not abrogated by pretreating the cells with an anti-CD18 mAb. MSUM stimulated more efficiently the adherence of neutrophils to non-preactivated hOB while CPPD were more efficient when hOB were preactivated. Crystal-free conditioned media from MSUM- or CPPD-stimulated hOB mobilized intracellular free calcium in human neutrophils. Thus, microcrystals were powerful promoters of neutrophil adherence to hOB via a CD18-independent mechanism. The bacterial peptide fMLP also stimulated the adherence of neutrophils to hOB. Functional neutrophil-hOB interactions could be important in bone pathophysiology of crystal- or infection-associated arthritis.     

Dual effects of formylpeptides on the adhesion of endotoxin-primed human neutrophils

Neutrophils, treated with sequential additions of bacterial products such as endotoxin (E. Coli lipopolysaccharide, LPS) and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), undergo to metabolic activation and express membrane-anchoring proteins that promote adhesion to serum-coated culture wells. By investigating the dose–response relationships of these phenomena, we have found that: (a) resting neutrophils do not produce a significant amount of superoxide (O) and show only minimal adhesion to serum-coated plastic surfaces; (b) fully activatory doeses (> 5 × 10^?8 M ) of fMLP induce the release of O and a significant increase of the cell adhesion; (c) pretreatment of the cells for 1 h with LPS augments cell adhesion to serum-coated culture wells in the absence of further stimulation and primes the neutrophils to enhanced fMLP-dependent O release; (d) addition of low, substimulatory doses of fMLP (from 10^?10 M to 5 × 10^?9 M ) inhibits and reverses the adhesion of LPS-treated cells, (e) high fMLP doses (> 10^?7 M ) are additive to LPS in promoting adhesion. Phorbol-myristate acetate (> 10^?9M) increased adhesion in both normal and LPS-treated neutrophils, but low doses of this stimulant did not inhibit adhesion. Low doses (10^?9 M ) of fMLP increased intracellular cyclic AMP in both normal and LPS-treated neutrophils, suggesting that stimulus-induced rises in cAMP may be the negative signal responsible for down-modulation of adhesion. Low (5 × 10^?9 M ) and high (5 × 10^?7 M ) fMLP doses induced the same increase of expression of CD11/CD18 integrins, indicating that the inhibition of adhesion caused by low doses is not due to quantitative down-regulation of integrins. These findings may provide an in vitro model of the complex biological events involved in the regulation of neutrophil adhesion.     

Involvement of Toll-like receptor 4 and Fc receptors gamma in human neutrophil priming by endotoxins from Escherichia coli

By using the fMLP-induced respiratory burst approach, the involvement of Toll-like receptor 4 (TLR4) in human neutrophil priming by S- or Re-glycoforms of endotoxin from Escherichia coli has been elucidated. The priming effect of Re-glycoform is more pronounced than that of the S-glycoform. Unexpectedly, fMLP-triggered generation of reactive oxygen species (ROS) by endotoxin primed neutrophils was amplified by preincubation of the cells with anti-TLR4 (HTA125) antibodies or with isotype-matched immunoglobulin IgG2a. The most significant finding of our study is that neutrophils exposed to anti-TLR4 antibodies retain their ability to distinguish between S- or Re-glycoforms being primed, respectively. Moreover, differentiated effect of HTA125 antibodies on functional responses of neutrophils during their priming and fMLP stimulation was revealed. Taking these results into consideration, it is reasonable to assume that there is a contribution of Fcγ receptors to fMLP-induced ROS generation by neutrophils preincubated with HTA125 or IgG2a and primed by endotoxins.     

Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.     

High mannose type N-linked oligosaccharides on endothelial cells may influence β2 integrin mediated neutrophil adherence in vitro

We report herein on the role of N-linked oligosaccharide processing of endothelial cell surface proteins on the adhesion of neutrophils. Monolayers of human umbilical vein endothelial cells were treated for 24 h with deoxymannojirimycin (DMJ), an inhibitor of golgi mannosidase I, which results in changes in glycoprotein processing, and then incubated with neutrophils to examine their ability to adhere to the treated endothelial cells. Treatment with DMJ, which leads to accumulation of high mannose type oligosaccharides, resulted in a twofold increase in adherence of phorbol ester (PMA) activated neutrophils compared to attachment to untreated endothelial cells. This adherence was likely mediated by the β2 integrin, Mac-1, and could be specifically inhibited with monoclonal antibodies to ICAM-1 and to the integrin β2 subunit. Similarly, IL-1 treatment resulted in a β2 integrin mediated increase in neutrophil adherence to the DMJ treated endothelial cells in a dose dependent manner. However, the IL-1 induced adherence was not significantly inhibited by the anti-ICAM-1 antibody, thus, suggesting the presence of other inducible components on the endothelial cell surface. Our results demonstrate that alterations in glycosylation of N-linked oligosaccharides, resulting in the synthesis of high mannose type sugars on molecules that may interact with the β2 integrins, leads to an increased adherence of PMA activated neutrophils to endothelial cells. © 1993 Wiley-Liss, Inc.     

Signaling functions of L-selectin in neutrophils: alterations in the cytoskeleton and colocalization with CD18.

Ligation and clustering of L-selectin by Ab ("cross-linking") or physiologic ligands results in activation of diverse responses that favor enhanced microvascular sequestration and emigration of neutrophils. The earliest responses include a rise in intracellular calcium, enhanced tyrosine phosphorylation, and activation of extracellular signal-regulated kinases. Additionally, cross-linking of L-selectin induces sustained shape change and activation of beta2 integrins, leading to neutrophil arrest under conditions of shear flow. In this report, we examined several possible mechanisms whereby transmembrane signals from L-selectin might contribute to an increase in the microvascular retention of neutrophils and enhanced efficiency of emigration. In human peripheral blood neutrophils, cross-linking of L-selectin induced alterations in cellular biophysical properties, including a decrease in cell deformability associated with F-actin assembly and redistribution, as well as enhanced adhesion of microspheres bound to beta2 integrins. L-selectin and the beta2 integrin became spatially colocalized as determined by confocal immunofluorescence microscopy and fluorescence resonance energy transfer. We conclude that intracellular signals from L-selectin may enhance the microvascular sequestration of neutrophils at sites of inflammation through a combination of cytoskeletal alterations leading to cell stiffening and an increase in adhesiveness mediated through alterations in beta2 integrins.     

Anti-inflammatory flavonoids and pterocarpanoid from Crotalaria pallida and C. assamica

One new isoflavone, 5,7,4'-trihydroxy-2'-methoxyisoflavone (3) and seven, and four known compounds were isolated from the barks of Crotalaria pallida and the seeds of C. assamica, respectively. The known compounds, apigenin (1) and 2'-hydroxygenistein (2), isolated from C. pallida, showed significant concentration-dependent inhibitory effects on the release of beta-glucuronidase and lysozyme from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC(50) values of 2.8+/-0.1 and 17.7+/-1.9, and 5.9+/-1.4 and 9.7+/-3.5 microM, respectively. The known compounds, daidzein (4) and 2'-hydroxydaidzein (6), isolated from C. pallida, inhibited of the release of lysozyme and beta-glucuronidase from rat neutrophils in response to fMLP/CB with IC(50) values of 26.3+/-5.5 and 13.7+/-2.6 microM, respectively. Compounds 1 and 4 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB with IC(50) values of 3.4+/-0.3 and 25.1+/-5.0 microM, respectively. Compounds 1 and 5, previously isolated from C. pallida, showed the inhibition of NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and LPS/interferon-gamma (IFN-gamma)-stimulated N9 microglial cells with IC(50) values of 10.7+/-0.1 and 13.9+/-1.1 microM, respectively. Flavonoids, suppressed chemical mediators in inflammatory cells, may have value in treatment and prevention of central and peripheral inflammatory diseases associated with excess production of chemical mediators.     

Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.