Desensitization of the human neutrophil degranulation response: Studies with 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid

The human polymorphonuclear neutrophil degranulation response to 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid was completely desensitized by preincubating the cells with small amounts of this same fatty acid. Desensitization developed within 1 min, persisted in thoroughly washed cells, and was not due to inactivation of the stimulus. These desensitized cells, however, degranulated partially in response to the ionophore A23187 and normally in response to C5a, N-formyl-methionyl-leucyl-phenylalanine, 1-$\text{0}\text{-}\text{alkyl-2-}\text{0}\text{-}\text{acetyl}\text{-}\text{sn}\text{-}\text{glycero-3-phosphocholine}$, and phorbol myristate acetate. Thus, the dihydroxy fatty acid is a unique stimulus which degranulates and desensitizes neutrophils by pathways at least partially distinct from those utilized by the other stimuli. The fatty acid, although rapidly formed in degranulating neutrophils, is unlikely to be an essential or universal mediator of the degranulation response.     

Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury

Summary A neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model substrate for elastases. To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up to 45 min. Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and lactate dehydrogenase (LDH). Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer, HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction. This effect could not satisfactorily be explained as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59% that of PPE. The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding cell layer; the value for HNE-solubilized protein was only 16%. Thus, the amount of elastin per microgram of solubilized protein for HNE was 15% that for PPE. Possible explanations for the greatly diminished elastolytic activity of HNE in the culture system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory fashion. Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that little loss of cells or cell viability occurred. The observed differences in elastolytic activity of HNE and PPE in the culture system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema. Supported by National Heart, Lung and Blood Institute, Bethesda, MD, grants NIH-HL-25229, HL-19717, and HL-33522. Presented in part at the April 1985 meeting of the Federation of American Societies for Experimental Biology.     

Diacylglycerols enhance human neutrophil degranulation responses: relevancy to a multiple mediator hypothesis of cell function

At 10 microM, 1-0-oleoyl-, 1-0-palmitoyl-, and 1-0-myristoyl-2-0-acetyl-glycerol weakly stimulated neutrophils to release lysozyme, an enzyme in secondary granules, but had no such effect on the release of a primary granule enzyme, beta-glucuronidase. The glycerides (1-10 microM) had a second effect on both granule populations: they enhanced the degranulating potencies of leukotriene B4, platelet-activating factor, a formylated oligopeptide, and C5a by 10- to 30-fold. In contrast, they were much less effective in enhancing responses to ionophore A23187 and partially inhibited responses to phorbol myristate acetate. The diether analogue, 1-0-hexadecyl-2-0-ethylglycerol was inactive in these regards. We suggest that diacylglycerols are a novel class of bioactive products mobilized from phosphoglycerides in stimulated neutrophils; as co-products of this mobilization, platelet-activating factor and leukotriene B4 may interact with diacylglycerols to promote cell function.     

Immunohistochemical analysis of inflammatory infiltrates around the injection site of a streptococcal preparation,OK-432, in rat tongue

On the assumption that neutrophils around the injection site of OK-432, a heat- and penicillin-treated lyophilized preparation of the Su strain ofStreptococcus pyogenes, enhance immunologic response through the production of Interleukin-1 (IL-1), OK-432 was injected into rat tongue, and specimens from the tongue were immunohistochemically investigated at various intervals after the injection, to clarify the process of inflammatory and immune responses at the injection site. Neutrophils and mononuclear cells appeared around the OK-432 injection site after 1 hour, increased to their maximum level at 24 hours, and then decreased from the 3rd to the 7th day. IL-1 was detected on neutrophils 3 hours after the injection, and OX-08-positive cells (suppressor/cytotoxic T cells and the majority of natural killer cells) remarkably increased. OX-39-positive cells (IL-2 receptor) appeared after 12 hours. These results suggest that neutrophils around the injection site of OK-432 at early phases of inflammation play a role in the expression of BRM function through IL-1.     

Human neutrophil interactions of a bispecific monoclonal antibody targeting tumor and human Fcγ RIII

 2B1 is a bispecific murine monoclonal antibody (bsmAb) targeting the c-erbB-2 and CD16 (FcγRIII) antigens. c-erbB-2 is over-expressed by a variety of adenocarcinomas, and CD16, the low-affinity Fcγ receptor for aggregated immunoglobulins, is expressed by polymorphonuclear leukocytes (PMN), natural killer (NK) cells and differentiated mononuclear phagocytes. 2B1 potentiates the in vitro lysis of c-erbB-2 over-expressing tumors by NK cells and macrophages. In this report, the interactions between 2B1 and PMN were investigated to assess the impact of these associations on in vitro 2B1-promoted tumor cytotoxicity by human NK cells. The peak binding of 2B1 to PMN was observed at a concentration of 10 μg/ml 2B1. However, 2B1 rapidly dissociated from PMN in vitro at 37°C in non-equilibrium conditions. This dissociation was not caused by CD16 shedding. When PMN were labeled with ¹²⁵I-2B1 and incubated at 37°C and the supernatants examined by HPLC analysis, the Fab regions of dissociated 2B1 were not complexed with shed CD16 extracellular domain. While most of the binding of 2B1 to PMN was solely attributable to Fab-directed binding to FcγRIII, PMN-associated 2B1 also bound through Fcγ-domain/FcγRII interactions. 2B1 did not promote in vitro PMN cytotoxicity against c-erbB-2-expressing SK-OV-3 tumor cells. When PMN were coincubated with peripheral blood lymphocytes, SK-OV-3 tumor and 2B1, the concentration of 2B1 required for maximal tumor lysis was lowered. Although PMN may serve as a significant competitive binding pool of systemically administered 2B1 in vivo, the therapeutic potential of the targeted cytotoxicity properties of this bsmAb should not be compromised. Received: 3 May 1995 / Accepted: 6 February 1996     

Sialidase Treatment Exposes the βT1-Integrin Active Ligand Binding Site on HL60 Cells and Increases Binding to Fibronectin

The migration of neutrophils from the circulation to areas of inflammation is the result of the sequential activation of multiple cellular adhesion molecules. βT1-Integrins are cell surface glycoproteins and the class of adhesion molecules responsible for binding to the extracellular matrix. The goal of this study was to determine the contribution of glycosylation, specifically the presence of sialic acid, to βT1-integrin adhesion in a neutrophil model. βT1-Integrins on differentiated HL60 cells were remodeled by treatment with the exoglycosidases, sialidase and βT-galactosidase. βT1-Integrin activity was determined by measuring adherence to the extracellular matrix protein fibronectin. The expression of βT1-integrins, βT2-integrins and activated βT1-integrins was determined by flow cytometry. Remodeling of βT1-integrins by treatment with sialidase increased adhesion by greater than 100%. Flow cytometric analysis of remodeled βT1-integrins demonstrated an increased expression of the activated βT1-integrin, but only minor increases in the expression of total βT1-and βT2-integrins. We postulate that glycosidase treatment increases adhesion and expression of activated βT1-integrins by exposure of the normally hidden ligand-binding site. The glycosylation of βT1-integrins on neutrophils may act to hide the ligand-binding site in unstimulated cells thereby contributing to the affinity modulation observed in neutrophil pl-integrin function.     

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF‐α‐primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)‐ and interleukin‐8 (IL‐8)‐induced NET formation exhibits a significant age‐related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL‐8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol‐12‐myristate‐13‐acetate (PMA) showed no age‐associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age‐related decline in NET and ROS generation. TNF‐α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age‐matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults.     

Estrogen modulates 7/4 antigen distribution within eccentrically contracted injured skeletal muscle

Abstract

Eccentric contractions are skeletal muscle stretches with concurrent active force production; these contractions commonly occur during dynamic sports activities and can cause acute muscle injury. Recovery from this injury depends in part on pro-inflammatory processes, such as neutrophil infiltration at the injured site, which is affected by estrogen. This estrogen effect has been examined broadly, but without distinguishing between major compartments within muscle in which neutrophil infiltration can occur. Therefore, we compared neutrophil antigen expression in two compartments of eccentrically contracted muscle of ovariectomized mice with or without estrogen. To quantify neutrophil antigen expression, serial cross sections of muscle were immunolabeled with antibodies that recognize 7/4 or Ly6C/G, then quantified using computer-assisted image analysis. At 48 h post injury, estrogen-positive (E+) mice had more 7/4-positive and Ly6C/G-positive myofibers, increased 7/4 area percentage, and more 7/4-positive cells in the connective tissue. In addition, E+ mice showed more 7/4-positive myofibers that were Ly6C/G-negative and more Ly6C/G-positive myofibers that were 7/4-negative. These data suggest that in injured muscle, estrogen increases 7/4 antigen in connective tissue and myofibers and is associated with more Ly6C/G-positive myofibers when the 7/4 antigen is absent from these myofibers.     

GPR35 promotes neutrophil recruitment in response to serotonin metabolite 5-HIAA

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The increasing effect of some synthetic peptides on luminol-dependent chemiluminescence of mouse blood

Some synthetic peptides increased the luminol-dependent chemiluminescence of mouse blood during phagocytosis. It is suggested that the levels of antimicrobial activity of the neutrophil peroxidase system can be raised very quickly (within some dozen of seconds) by these peptides. This raises the possibility of finding a new approach to the therapy for infectious diseases.