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PubMed1988

Preincubation of bovine blood neutrophils with bovine herpesvirus-1 does not impair neutrophil interaction with Pasteurella haemolytica A1 in vitro.

In this study we examined the direct effects of bovine herpesvirus-1 on the interaction of bovine blood neutrophils with Pasteurella haemolytica A1. Preincubation of neutrophils for approximately 2 h in vitro with BHV-1 at a multiplicity of infection of 5:1 had no effect on neutrophil random migration and directed migration to zymosan-activated bovine serum. Neutrophils also were unimpaired in their ability to ingest and kill P. haemolytica A1. Preincubation of neutrophils with BHV-1 did not elicit an oxidative burst, as measured by luminol-enhanced chemiluminescence, nor did it alter neutrophil chemiluminescence in response to opsonized P. haemolytica A1. Prolonged preincubation with BHV-1 for 18-24 h similarly did not affect neutrophil chemiluminescence in response to opsonized P. haemolytica A1. The susceptibility of neutrophils to the lethal effects of crude P. haemolytica cytotoxin also was unaltered by preincubation with BHV-1. We observed no evidence of BHV-1 replication in bovine neutrophils as determined by indirect immunofluorescence and electron microscopy. Previous reports have indicated that active BHV-1 infection alters certain neutrophil functions and results in hypersusceptibility to pulmonary pasteurellosis. Our results suggest that these effects are unlikely to be mediated directly by BHV-1, but instead may reflect the action of endogenous mediators that are released during active BHV-1 infection.

Animals↗
PubMed1991

Adherence to bovine neutrophils and suppression of neutrophil chemiluminescence by Mycoplasma bovis.

The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.

Animals↗
PubMed1992

A model for the recruitment of neutrophils at sites of inflammation. Physiological relevance of in vivo neutrophil aggregation.

Adhesive glycoproteins on both neutrophils and vascular endothelial cells are known to mediate adhesive interaction between the two cell types. We propose that activation of endothelial cells will lead to the capture of unactivated neutrophils, localizing them at inflammatory sites. The interaction between activated endothelium and captured neutrophils will result in the stimulation of adherent neutrophils. Adherent activated neutrophils are then able to recruit incoming unactivated neutrophils by capturing them, further increasing the number of neutrophils at the inflammatory site. The formation of an adherent neutrophil aggregate will reduce plasma leakage from the vasculature as neutrophils migrate into tissue and will protect migrating neutrophils from shear stress of blood flow.

Animals↗
PubMed2006

Differential expression analysis of proteins from neutrophils in the periparturient period and neutrophils from dexamethasone-treated dairy cows.

Neutrophils play an important role in the host immune system's defense against pathogens. It has been established that neutrophil functionality is suppressed in dairy cows at parturition. The periparturient immunosuppression seen in dairy cattle is associated with an increase in the incidence of mastitis. Using amine-reactive isobaric tagging reagents we have measured relative protein expression from normal prepartum neutrophils and neutrophils isolated during immunosuppression at parturition. We found over 40 proteins that are differentially expressed at parturition compared to prepartum. In addition, we measured relative protein expression from normal neutrophils and neutrophils obtained from cows treated with an immunosuppressive dose of dexamethasone. We found over 70 proteins are differentially expressed during dexamethasone treatment. We then compared protein expression changes in dexamethasone-induced immunosuppression to periparturient immunosuppression. A number of proteins underwent similar expression changes in both dexamethasone and periparturient immunosuppressed neutrophils. Most significantly, we found a significant number of proteins whose relative expression was not the same for these two different conditions that cause neutrophil dysfunction. The data demonstrates that there are both similarities and differences in neutrophil protein expression in the naturally occurring immunosuppression observed at parturition compared to dexamethasone-induced immunosuppression in the bovine neutrophil.

Animals↗
PubMed1987

NADPH-binding component of the superoxide-generating oxidase in unstimulated neutrophils and the neutrophils from the patients with chronic granulomatous disease.

The NADPH-binding component of the neutrophil superoxide-generating oxidase was studied in the particulate oxidase fractions obtained from the neutrophils of normal and chronic-granulomatous-disease (CGD) patients. The molecular mass of the NADPH-binding component of the stimulated human neutrophils, which was labelled with the 2',3'-dialdehyde derivative of NADPH and sodium cyanoboro[3H]hydride, was 66 kDa. The 66 kDa component was also labelled in monocytes, but not in red blood cells, platelets and lymphocytes. The particulate oxidase fractions obtained from the patients with CGD had a diminished amount of FAD, whether they contained cytochrome b558 or not. The fractions labelled with the NADPH analogue showed that CGD patients had the NADPH-binding component in the neutrophils. The molecular mass of the component was identical with that of the normal neutrophils. The patients are thought to have an intact NADPH-binding domain of the oxidase in the neutrophils in spite of a diminished amount of FAD in the particulate fractions. The component of the oxidase in the resting neutrophils was also labelled with the analogue. The molecular mass of the component in the resting neutrophils was identical with that of the stimulated neutrophils, and the component was not phosphorylated during the activation process. These results indicate that the NADPH-binding component of the oxidase, which is specific to phagocytes, is present in the resting neutrophils and that the component does not change with respect to molecular mass during the activation process.

Cytochrome b Group↗
PubMed2000

Target antigens for anti-neutrophil cytoplasmic autoantibodies (ANCA) are on the surface of primed and apoptotic but not unstimulated neutrophils.

The reaction of ANCA with ANCA antigens on the surface of neutrophils may play a critical role in the pathogenesis of ANCA vasculitis. Therefore, an understanding of the circumstances that result in surface expression of these antigens is important for an understanding of pathogenic mechanisms. In this study we investigated the surface expression of ANCA antigens on quiescent, primed, and apoptotic neutrophils. ANCA antigens and other granule constituents were not detected on the surface of neutrophils in freshly heparinized blood. ANCA antigens were on the surface of neutrophils primed by in vitro incubation for 4 h and 8 h. These cells did not show evidence of apoptosis. After 24 h incubation, about 30% of the neutrophils were apoptotic, and ANCA antigens and other granule constituents were present on the surface of both apoptotic and non-apoptotic cells. Our data indicate that there are no ANCA antigens on the surface of quiescent neutrophils, but that they are on the surface of primed neutrophils before the cells become apoptotic, and remain on the surface of cells after they become apoptotic. Based on these observations, we hypothesize that ANCA can react in vivo with primed but not quiescent neutrophils. Previously published observations indicate that the interaction of ANCA with primed neutrophils results in neutrophil activation, which may be involved in the pathogenesis of ANCA vasculitis.

Animals↗
PubMedDate not supplied

Neutrophil collection using modified fluid gelatin. Effect on in vitro and in vivo neutrophil function.

Modified fluid gelatin (MFG) is a possible alternative to hydroxyethyl starch (HES) for use as a red cell sedimenting agent in neutrophil collection procedures. Since the efficacy of neutrophil transfusion therapy depends upon the integrity of the infused cells, we examined the in vitro and in vivo function of neutrophils collecting using MFG. Neutrophils were collected from 17 normal subjects by continuous flow centrifugation using MFG in place of HES. In vitro measurement of neutrophil function included dye exclusion, phagocytosis, candidacidal activity, staphylococcal killing, chemotaxis, and random migration. For in vivo studies, neutrophils were labeled with 3H-diisopropylfluorophosphate, reinfused into the donor, and blood kinetics and skin chamber accumulation of the labeled cells were measured. In vivo results were compared with results from previous studies using neutrophils collected with HES or by phlebotomy. MFG neutrophils were normal by all in vitro functional criteria and localized normally at an in vivo inflammatory site. Intravascular survival (T 1/2 = 3.3 +/- 0.9 hours) was significantly shorter than normal (T 1/2 = 7.3 +/- 2.3 hours) but was identical to that of cells collected using HES. Thus, on the basis of neutrophil functional capabilities, MFG appears to be an acceptable alternative to HES in collection of neutrophils for transfusion.

Cell Separation↗
PubMed1993

Analysis of the expression of neutrophil-specific antigen NB1: characterization of neutrophils that react with but are not agglutinated by anti-NB1.

The neutrophil-specific NB antigen system has been serologically characterized with human alloantisera. Two alleles, NB1 and NB2, have been described. NB1 is expressed on a subpopulation of peripheral blood neutrophils in 97 percent of healthy donors. Human alloantibodies have been used to identify the 58- to 64-kDa glycoprotein (GP) on which NB1 is located. NB1 can usually be detected by both a granulocyte immunofluorescence (GIF) assay and a granulocyte agglutination (GA) assay, but neutrophils from some donors have been found to react with anti-NB1 in GIF but not in GA assays. To determine if the latter neutrophils express NB1 and the corresponding 58- to 64-kDa GP, these neutrophils were probed with rabbit and human sera specific for NB1. First, the proportion of neutrophils that express NB1 was quantitated. Neutrophils from donors that typed as NB1-positive in both GA and GIF assays were analyzed by flow cytometry with antisera to NB1. Human and rabbit anti-NB1 reacted with 71 +/- 17 percent and 70 +/- 17 percent of neutrophils, respectively. There was no difference in the expression of NB1 in NB1-homozygous and NB1-heterozygous individuals. In contrast, significantly fewer neutrophils from four donors that typed as NB1-positive in GIF assay but not GA assay reacted with human (27 +/- 12%; p < 0.001) and rabbit (26 +/- 12%; p < 0.001) anti-NB1. When neutrophils from these same four donors were probed with rabbit and human anti-NB1 by immunoblotting and immunoprecipitation, the 58- to 64-kDa GP was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Isolation of cDNA encoding guinea pig neutrophil cationic antibacterial polypeptide of 11 kDa (CAP11) and evaluation of CAP11 mRNA expression during neutrophil maturation.

Neutrophils contain various antibacterial polypeptides and proteins in the granules that contribute to the killing of microorganisms. Recently, we have purified a cationic antibacterial polypeptide of 11 kDa (CAP11) from guinea pig neutrophil granules. CAP11 is a homodimer of G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43 joined with one disulfide bond. In this study, to understand the regulation of CAP11 expression, we isolated and analyzed cDNA encoding CAP11. Furthermore, we investigated the expression of CAP11 mRNA during neutrophil maturation and localization of CAP11 among neutrophil granule subsets. Sequence analysis of CAP11 cDNA isolated from guinea pig bone marrow cells using rapid amplification of cDNA ends technique indicated that CAP11 is synthesized as a precursor comprising 178 amino acid residues, which is composed of a signal peptide (N-terminal 29 residues), a propeptide (106 residues), and a C-terminal mature peptide (43 residues). Interestingly, the predicted CAP11 precursor displayed the characteristic features of cathelicidins, a novel protein family of antibacterial polypeptides with a conserved cathelin-like pro-region and a variable C-terminal antibacterial domain. Northern blot and Western blot analyses using neutrophils, macrophages, eosinophils, mononuclear cells, and bone marrow cells revealed that only neutrophils and bone marrow cells expressed CAP11 mRNA and contained CAP11, suggesting that expression of CAP11 is neutrophil lineage-specific. Furthermore, Northern blot analysis using bone marrow cells separated according to their maturation stages showed that CAP11 mRNA was predominantly expressed in the cells at later stages of neutrophil maturation. Consistent with this, in situ hybridization using CAP11-specific cRNA probe demonstrated that CAP11 mRNA was primarily expressed at metamyelocyte stage. In addition, extracellular release assay revealed that CAP11 was readily released from neutrophils accompanied with gelatinase by low concentrations of N-formyl-Met-Leu-Phe without release of specific and azurophil granule components, and CAP11 was found to be exclusively present in the fraction containing gelatinase granules, prepared by Percoll density gradient centrifugation. Together these observations indicate that CAP11 is a member of cathelicidin family and its mRNA is preferentially expressed at the later stage of neutrophil maturation (i.e. metamyelocyte stage). Furthermore, CAP11 may be stored in the granule subset, possibly the gelatinase granule.

Amino Acid Sequence↗
PubMed1995

Effect of N-3 polyunsaturated fatty acid supplemented diet on neutrophil-mediated ileal permeability and neutrophil function in the rat.

RATIONALE AND OBJECTIVE: Fish oil, rich in n-3 polyunsaturated fatty acids, can alter leukotriene production and hence neutrophil function, factors which may be important in the inflammation of Crohn's disease (CD). Therefore we studied the effect of dietary PUFA on neutrophil mediated ileal inflammation and neutrophil function in the rat. METHODS: Animals were ad libitum-fed pellet diets containing 9.5% fish oil (menhaden oil, rich in n-3 PUFA) with 0.5% safflower oil, 10% safflower oil (rich in n-6 PUFA) or standard chow (6% fat) for 50 days prior to the study. Weight and circulating leukocyte and total neutrophil counts were identical in all three groups. Neutrophil mediated ileal inflammation induced by formyl-methionyl-leucyl-phenylalanine (fMLP) perfusion was evaluated by measuring macromolecular uptake of radiolabelled dextran (MW 70,000) and changes in mucosal neutrophil infiltration. RESULTS: The fish oil diet group showed no difference in fMLP-induced permeability changes relative to the Chow Control group. However, the Safflower Oil supplemented diet group had a reduced permeability response (p < 0.01). Mirroring the permeability changes, there was diminished mucosal neutrophil infiltration in the Safflower Oil group following ileal perfusion with fMLP (< .005). Chemotaxis and chemiluminescence, two important neutrophil functions, were also significantly suppressed in the Safflower Oil animals (p < 0.05). CONCLUSIONS: The failure of a n-3 PUFA enriched diet to diminish the ileal inflammatory response to a bacterial peptide and suppress neutrophil function in the rat suggests such therapy would not be expected to be highly successful in CD. However, it requires confirmation in man, especially under the more complicated inflammatory conditions found in CD. On the other hand, the decreased neutrophil mediated responses with a high linoleic acid (n-6 PUFA) diet warrant further investigation.

Animals↗
PubMed2006

Both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary Streptococcus pneumoniae infection.

Since secondary Streptococcus pneumoniae infections greatly increase the mortality of influenza infections, we determined the relative roles of neutrophil-dependent and -independent mechanisms in increased susceptibility to S. pneumoniae during influenza infection. Mice infected with influenza for 6 days, but not 3 days, showed a significant increase in susceptibility to S. pneumoniae infection compared to mice not infected with influenza. There was significant neutrophil accumulation in the lungs of S. pneumoniae-infected mice regardless of whether or not they were infected with influenza for 3 or 6 days. Depletion of neutrophils in these mice resulted in increased susceptibility to S. pneumoniae in both the non-influenza-infected mice and mice infected with influenza for 3 days but not in the mice infected with influenza for 6 days, indicating that a prior influenza infection of 6 days may compromise neutrophil function, resulting in increased susceptibility to a S. pneumoniae infection. Neutrophils from the lungs of mice infected with influenza for 3 or 6 days exhibited functional impairment in the form of decreased phagocytosis and intracellular reactive oxygen species generation in response to S. pneumoniae. In addition, neutrophil-depleted mice infected with influenza for 6 days were more susceptible to S. pneumoniae than neutrophil-depleted mice not infected with influenza, indicating that neutrophil-independent mechanisms also contribute to influenza-induced increased susceptibility to S. pneumoniae. Pulmonary interleukin-10 levels were increased in coinfected mice infected with influenza for 6 days but not 3 days. Thus, an influenza infection of 6 days increases susceptibility to S. pneumoniae by both suppression of neutrophil function and by neutrophil-independent mechanisms such as enhanced cytokine production.

Animals↗
PubMed1986

Neutrophil activity in abscess-bearing mice: comparative studies with neutrophils isolated from peripheral blood, elicited peritoneal exudates, and abscesses.

Intraabdominal abscesses were induced in mice by intraperitoneal inoculation of Bacteroides fragilis and Escherichia coli plus bran as the abscess-potentiating agent. Six- or seven-day-old abscesses were mechanically disaggregated in buffer, and the cells obtained were fractionated on discontinuous Percoll density gradients. Neutrophil populations of different density, each approximately 90% pure, were isolated. When the abscess-derived neutrophils were subsequently incubated with normal serum in vitro under aerobic conditions, the viability of the gram-negative bacteria that had been phagocytosed within the abscess did not change significantly. This anergy to intracellular bacteria (on subsequent incubation in vitro under optimal conditions for phagocytic killing) was also found for neutrophils that had been obtained from abscesses induced by a mixture that included Proteus mirabilis plus B. fragilis and from those induced by E. coli plus P. mirabilis. While unable to significantly kill intracellular organisms that had been phagocytosed in vivo, the abscess-derived neutrophils could engulf and kill organisms to which they were exposed in vitro. Neutrophils from abscesses induced by P. mirabilis only plus bran killed that organism introduced in vitro significantly more effectively than the organisms that had been engulfed in vivo. In contrast, neutrophils from abscesses induced by the gram-positive organism Staphylococcus aureus plus bran were able to kill their intracellular organisms on subsequent incubation in vitro as effectively as they could kill added S. aureus. Neutrophils isolated from the peripheral blood and from induced peritoneal exudates of abscess-bearing mice were able to phagocytose and kill organisms in vitro with greater efficiency than abscess-derived neutrophils. The mechanism whereby neutrophils from abscesses induced by the gram-positive organism S. aureus can kill the organisms phagocytosed in vivo on subsequent in vitro incubation, in contrast to the relative anergy to their intracellular organisms displayed by neutrophils derived from abscesses induced by combinations of gram-negative bacteria, is not known.

Abscess↗
PubMed2003

Deficiency in neutrophil elastase does not impair neutrophil recruitment to inflamed sites.

To reach the sites of inflammation, neutrophils traverse the endothelium, its underlying basement membrane, and other barriers depending on the localization of the insulting agent. Whether neutrophil elastase (NE) plays a role in neutrophil recruitment to inflamed sites is still debatable. By exploiting mice deficient in NE (NE(-/-)), we sought to address this dilemma. We recruited neutrophils to the lungs or the peritoneum of wild-type (WT) or NE(-/-) mice by intranasal or intraperitoneal challenge with Pseudomonas aeruginosa or its lipopolysaccharide. At designated times post-inoculation (0, 4, 24, and 48 h), groups of mice were killed to assess changes in leukocyte counts and inflammatory responses. NE(-/-) and WT mice had normal circulating leukocyte numbers including neutrophils and changes in the hemograms in the setting of acute inflammation were indistinguishable. Analyses of lung tissues or fluids from the lungs and peritoneum found that regardless of the inflammatory model, the leukocyte counts including neutrophils and the inflammatory response were similar in NE(-/-) and WT mice at all time points. In vitro, neutrophils isolated from the lungs or the peritoneum of NE(-/-) and WT mice had comparable chemotactic and respiratory-burst functions and migrated normally through Matrigel in response to various stimuli. Interestingly, preincubation of human peripheral blood neutrophils with NE physiologic inhibitors did not alter the migration of the cells through Matrigel. In sum, our findings present the first in vivo description that the absence of NE does not impair neutrophil recruitment to inflamed sites and that NE is not required for basement membrane transmigration of neutrophils.

Administration, Intranasal↗
PubMed2005

Induction of human airway smooth muscle apoptosis by neutrophils and neutrophil elastase.

Neutrophils are an important component of airway inflammation and may interact with human airway smooth muscle cells (HASMC). We investigated the effect of neutrophils and of neutrophil-derived proteases on HASMC survival. When co-incubated with neutrophils (0.1-1 x 10(6) cells/ml), attachment of human ASMC was reduced to 12.3 +/- 4.3% compared with untreated controls after 72 h. HASMC showed nuclear condensation and fragmentation (41.6 +/- 8.1% compared with baseline of 3.1 +/- 0.4%), and the biochemical markers of apoptosis, annexin V binding (9.7 +/- 0.7%; baseline 1.1 +/- 0.3%) and cleaved caspase-3 expression, were observed. The proteolytic activity released by neutrophils was essential for the proapoptotic effect because inhibition of elastase activity by alpha(1)-antitrypsin and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) reduced HASMC apoptosis. Human neutrophil elastase (0.1-3 microg/ml) induced apoptosis of HASMC, as well as other neutrophil serine proteases, cathepsin G, and proteinase 3. Fibronectin degradation products were present in HASMC supernatants exposed to neutrophil-conditioned media and to neutrophil elastase. The local release of proteases from neutrophils present in airway smooth muscle cells may lead to HASMC apoptosis as a result of matrix degradation and loss of cell attachment. This may limit pathologic changes such as ASMC hyperplasia and extracellular matrix deposition seen in airway remodeling.

Apoptosis↗
PubMed1986

Neutrophil adhesion abnormality with deficient surface membrane proteins (gp 110 and p 98): the effect of their antibodies on the function of normal neutrophils.

A sister and brother previously described with neutrophil adhesion defects and a lack of two neutrophil membrane proteins, glycoprotein with a molecular weight of 110 K and 115 K, were further studied. Rabbit polyclonal IgG antibodies were raised against neutrophil membrane proteins of approximately 110 K from normal individuals, and absorbed with the siblings' neutrophil membrane proteins. The antibodies thus absorbed reacted with two normal neutrophil membrane proteins, a 110 K glycoprotein and a 98 K protein, which were totally deficient in the siblings' neutrophils. The polyclonal antibodies were also reactive with a 95 K membrane protein of normal lymphocytes. The protein was missing in the siblings' lymphocytes. Three membrane proteins, Mac-1, LFA-1, and p 150, 95 each consisting of a specific alpha-subunit and a beta-subunit common to them, have been reported to be deficient in neutrophils from several patients with a neutrophil adhesion disease. Using monoclonal antibodies to Mac-1 alpha, LFA-1 alpha, and to the beta-subunit, it was deduced that the siblings' neutrophils lacked the above three membrane proteins. The disease in the siblings is thus identical with that previously described. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of immunoprecipitates, formed by reacting labeled normal neutrophil membrane proteins with our polyclonal antibodies, revealed three bands, almost identical with that of immunoprecipitates formed with the anti-beta-subunit monoclonal antibody. This finding indicates that the polyclonal antibodies reacted mainly with the beta-subunit of the membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2000

Decreased neutrophil adhesion to human cytomegalovirus-infected retinal pigment epithelial cells is mediated by virus-induced up-regulation of Fas ligand independent of neutrophil apoptosis.

Human CMV (HCMV) retinitis frequently leads to blindness in iatrogenically immunosuppressed patients and in the end stage of AIDS. Despite the general proinflammatory potential of HCMV, virus infection is associated with a rather mild cellular inflammatory response in the retina. To investigate this phenomenon, the influence of HCMV (strains AD169 or Hi91) infection on C-X-C chemokine secretion, ICAM-1 expression, and neutrophil recruitment in cultured human retinal pigment epithelial (RPE) cells was studied. Supernatants from infected cultures contained enhanced levels of IL-8 and melanoma growth-stimulating activity/Gro alpha and induced neutrophil chemotaxis compared with supernatants from uninfected RPE cells. Despite HCMV-induced ICAM-1 expression on RPE cells, binding of activated neutrophils to HCMV-infected RPE cells and subsequent transepithelial penetration were significantly reduced. Reduced neutrophil adhesion to infected RPE cells correlated with HCMV-induced up-regulation of constitutive Fas ligand (FasL) expression. Functional blocking of FasL on RPE cells with the neutralizing mAbs NOK-1 and NOK-2 or of the Fas receptor on neutrophils with mAbB-D29 prevented the HCMV-induced impairment of neutrophil/RPE interactions. Fas-FasL-dependent impairment of neutrophil binding had occurred by 10 min after neutrophil/RPE coculture without apoptotic signs. Neutrophil apoptosis was first detected after 4 h. Treatment of neutrophils with a specific inhibitor of caspase-8 suppressed apoptosis, whereas it did not prevent impaired neutrophil binding to infected RPE. The current results suggest a novel role for FasL in the RPE regulation of neutrophil binding. This may be an important feature of virus escape mechanisms and for sustaining the immune-privileged character of the retina during HCMV ocular infection.

Adult↗
PubMed2005

Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils.

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of alpha1-protease inhibitor (alpha1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of alpha1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of alpha1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of alpha1-Pi. HNE was not released from the cell surface by oxidized, inactive alpha1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by alpha1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.

Enzyme Stability↗
PubMed1989

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen.

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: 1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; 2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and 3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules.

Animals↗