Induction of cyclo-oxygenase synthesis in human promyelocytic leukaemia (HL-60) cells during monocytic or granulocytic differentiation.

Cyclo-oxygenase (COX) production in human promyelocytic leukaemia (HL-60) cells was studied during monocytic differentiation induced by 1 alpha, 25-dihydroxyvitamin D3 (24 nM; 3 days) or phorbol 12-myristate 13-acetate (100 nM; 1 day), or during granulocytic differentiation induced by retinoic acid (1 microns; 4 days). Undifferentiated or differentiated HL-60 cells were labelled with [35S]methionine, and membrane-bound COX was solubilized and quantified by SDS/PAGE. Immunoprecipitated 35S-labelled COX from cells induced to differentiate into monocytic or granulocytic lineage were clearly detected on the autoradiograms as a protein of approx. 70 kDa molecular size, whereas only a very faint COX band was detected in untreated HL-60 cells. During both monocytic and granulocytic differentiation, COX activity (measured by the conversion of exogenous arachidonic acid into prostaglandin E2) was dramatically increased. In addition, thromboxane synthesis was preferentially enhanced during monocytic differentiation. HL-60 cells, induced to differentiate into the monocytic or granulocytic lineage, provide a useful tool to investigate the cellular mechanisms involved in regulation of the synthesis of individual prostanoid-metabolizing enzymes.     

Tumor necrosis factor-alpha is induced through phorbol ester--and glycated human albumin-dependent pathway in THP-1 cells

Tumor necrosis factor-alpha (TNF-alpha) is involved in insulin resistance. Since the fact that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit the induction of TNF-alpha by phorbol ester, but not by lipopolysaccharide (LPS), suggests two pathways to induce TNF-alpha, we investigated the mechanisms of glycated human albumin (GHA)- or phorbol ester-induced TNF-alpha in THP-1 cells. GHA induced TNF-alpha release in differentiated THP-1 cells, while phorbol ester induced TNF-alpha release in undifferentiated cells but did not induce TNF-alpha in differentiated cells. Forskolin (adenylate cyclase activator) affected more the GHA-induced TNF-alpha release than the phorbol 12-myristate 13-acetate (PMA)-induced one in undifferentiated cells. Staurosporine [protein kinase-C (PK-C) inhibitor] and PD98059 [mitogen-activated protein kinase inhibitor (MAPK)] only partially inhibited GHA-induced TNF-alpha. Catalase completely inhibited GHA-induced TNF-alpha release; however, superoxide dismutase (SOD) had no effect. These results suggest at least two pathways to induce TNF-alpha (phorbol ester- and GHA-dependent ways) and that GHA-induced TNF-alpha release is through predominantly catalase-dependent way in differentiated THP-1 cells.     

Increase in chemiluminescence induced by receptor-independent stimulation of polymorphonuclear cells from psoriatic patients

Using a lucigenin-dependent chemiluminescence the authors have measured the "respiratory burst" in polymorphonuclear cells from psoriatic patients and controls. Measurements were performed under stimulation with zymosan, phorbol 12-myristate 13-acetate and latex beads. It has been revealed that the response after stimulation with zymosan increased although there was no significant difference between patients and controls, while the response after stimulation with phorbol 12-myristate 13-acetate and latex beads was significantly increased. Our results suggest an enhanced metabolic activity of polymorphonuclear cells induced by stimuli acting independently from cell-membrane receptors. Therefore an enhanced excitability of polymorphonuclear leukocytes of psoriatic patients is supposed.     

A correlation between phorbol diester-induced protein phosphorylation and superoxide anion generation in HL-60 cells during granulocytic maturation

As HL-60 cells matured along the granulocytic pathway, phorbol diester-induced superoxide anion production was compared to phorbol diester-induced protein phosphorylation using an in vitro phosphorylation technique. Maturation was induced by 0, 2, 4, or 6 days incubation with dimethyl sulfoxide (Me2SO). In 0 day Me2SO HL-60 cells, phorbol 12-myristate 13-acetate induced phosphorylation of protein pp29 (Mr = 28,600) and to a lesser extent protein pp76 (Mr = 76,300). With increased time of Me2SO incubation, phorbol 12-myristate 13-acetate induced phosphorylation of pp212 (Mr = 211,800), pp134 (Mr = 134,200), and pp76, whereas the phosphorylation of pp29 did not change appreciably. In close agreement with this increase in protein phosphorylation was the observed increase in phorbol diester-induced superoxide anion formation. Morphological characterization of cells during Me2SO-induced differentiation reveals that these increases in phorbol diester responses are probably attributable to the proportional rise in metamyelocytes, band, and segmented neutrophils. A variety of phorbol diesters increased superoxide anion generation in HL-60 cells differentiated into granulocyte-like cells by 6-day incubation with Me2SO. The structure-activity relationship of these phorbol diester derivatives for protein phosphorylation was strongly correlated to their ability to increase superoxide anion generation. Thus, we propose that phorbol diester-induced phosphorylation of pp212, pp134, and pp76, but not pp29 may play a role in mediating the functional response of phorbol diester-induced superoxide anion generation in HL-60 cells differentiated into mature granulocyte-like cells.     

Regulation of 5-oxo-ETE synthesis by nitric oxide in human polymorphonuclear leucocytes upon their interaction with zymosan and Salmonella typhimurium

In the present study we have presented data on the regulation of LT (leukotriene) and 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) syntheses in human neutrophils upon interaction with OZ (opsonized zymosan) or Salmonella typhimurium. Priming of neutrophils with PMA (phorbol 12-myristate 13-acetate) and LPS (lipopolysaccharide) elicits 5-oxo-ETE formation in neutrophils exposed to OZ, and the addition of AA (arachidonic acid) significantly increases 5-oxo-ETE synthesis. We found that NO (nitric oxide)-releasing compounds induce 5-oxo-ETE synthesis in neutrophils treated with OZ or S. typhimurium. Exposure of neutrophils to zymosan or bacteria in the presence of the NO donor DEA NONOate (1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium) considerably increased the conversion of endogenously formed 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) to 5-oxo-ETE. To our knowledge, this study is the first to demonstrate that NO is a potent regulator of 5-oxo-ETE synthesis in human polymorphonuclear leucocytes exposed to Salmonella typhimurium and zymosan.     

The relationship between lysosomal enzyme release and protein phosphorylation in human monocytes stimulated by phorbol esters and opsonized zymosan

Since it was established that phorbol esters bind to and activate protein kinase C, a proposed mechanism of action for these compounds has been the phosphorylation of specific protein substrates (Niedel, J. E., Kuhn, L. J., and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 36-40; Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851). To better understand this proposed relationship, we investigated the ability of a series of phorbol esters to elicit lysosomal enzyme release (LER) and specific substrate phosphorylation in human monocytes. In this system, phorbol 12-myristate 13-acetate stimulated the secretion of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase in both a time- and concentration-dependent manner. Furthermore, the ability of a series of phorbol esters to stimulate LER was characterized and found to be in good agreement with the relative order of these compounds to stimulate the phosphorylation of four endogenous protein substrates. Phorbol ester-stimulated protein phosphorylation was examined in intact cell preparations and found to be concentration and structure-dependent. The phosphoproteins (pp) were designated pp28, pp55, pp61, and pp66 corresponding to their molecular masses in kilodaltons. These findings are consistent with the hypothesis that phorbol ester-mediated effects are the result of protein kinase C activation and subsequent protein phosphorylation. Finally, opsonized zymosan was found to elicit a concentration-dependent stimulation of N-acetyl-beta-D-glucosaminidase release similar in magnitude and time course to phorbol ester-stimulated LER. Opsonized zymosan also stimulated the phosphorylation of two phosphoproteins (pp61 and pp66) in a concentration-dependent manner. Specific phosphorylation of pp61 and pp66 by both secretagogues, phorbol 12-myristate 13-acetate and opsonized zymosan, suggests these two proteins may be key to the functional response of LER in human monocytes.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Activated human eosinophils synthesize new proteins

In order to study the biochemical consequences of prolonged in vitro activation of human blood eosinophils, aqueous whole cell lysates, cell-free supernatants from resting eosinophils, and cells activated with opsonized zymosan, calcium ionophore (A23187), N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and phorbol 12-myristate 13-acetate (PMA) were analyzed by polyacrylamide gel electrophoresis (PAGE). In comparison to resting eosinophils, opsonized zymosan-activated eosinophil extracts demonstrated altered protein composition on both the native PAGE and sodium dodecyl sulfate (SDS) -PAGE. Three new polypeptides of apparent molecular mass 24 kDa, 43 kDa and 60 kDa appeared on SDS-PAGE gels when opsonized zymosan-activated eosinophil extracts were electrophoresed. In contrast, extracts from fMet-Leu-Phe, A23187, and PMA-activated eosinophils demonstrated neither altered polypeptide composition nor new polypeptides. Opsonized zymosan also induced the incorporation of L-[35S]methionine into eosinophil proteins and this was completely blocked by pretreating the cells with cycloheximide. This finding suggests that eosinophils activated by certain stimuli synthesize new proteins. These newly synthesized proteins, which are freely secreted into the medium during cell activation, may possess important immunological functions.     

Essential requirement of cytosolic phospholipase A(2) for stimulation of NADPH oxidase-associated diaphorase activity in granulocyte-like cells.

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA(2) in regulation of NADPH oxidase-associated diaphorase activity. A novel diaphorase activity assay, using 4-iodonitrotetrazolium violet as an electron acceptor, was used in permeabilized neutrophils and PLB-985 cells differentiated toward the granulocytic or monocytic phenotypes. Phorbol 12-myristate 13-acetate, guanosine 5'-3-O- (thio)triphosphate (GTP gamma S), or FMLP stimulated a similar diphenylene iodonium-sensitive diaphorase activity pattern in neutrophils and in differentiated parent PLB-985 cells. This diaphorase activity was not detected in undifferentiated cells, but developed during differentiation. Furthermore, diaphorase activity could not be stimulated in permeabilized neutrophils from X-linked CGD patients and in differentiated gp91(phox)-targeted PLB-985 cells that lacked normal expression of gp91(phox), but was restored to these cells following transduction with retrovirus encoding gp91(phox). The differentiated PLB-D cells showed no diaphorase activity when stimulated by either GTP gamma S or FMLP, and only partial activation when stimulated with phorbol 12-myristate 13-acetate. Diaphorase activity in response to either agonists was fully restored by the addition of 10 microm free AA. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.     

Signal transduction in endotoxin-stimulated synthesis of TNF-alpha and prostaglandin E2 by rat Kupffer cells. Role of extracellular calcium ions and protein kinase C.

Endotoxin is a well established elicitor of cytokine production in mononuclear cells. Nevertheless, the path of signal transduction between the crucial contact of the cells with endotoxin (lipopolysaccharide) and the synthesis and release of the mediators is yet poorly understood. In particular, the involvement of Ca2+ and protein kinase C in this process is still a matter of controversy. Here, it will be demonstrated that removal of extracellular Ca2+ by EGTA does not have a significant effect on the endotoxin-stimulated production of tumor necrosis factor-alpha (TNF-alpha) and on total protein synthesis in rat Kupffer cells. However, the release of prostaglandin E2 could not be raised above the basal level under these conditions. Treatment with inhibitors of protein kinase C such as the isoquinoline derivative, H-7, or staurosporin is without influence on TNF-alpha synthesis. The depletion of protein kinase C through preincubation of rat Kupffer cells with phorbol 12-myristate 13-acetate for 24 h was also without effect on TNF-alpha production. The effectiveness of these inhibitors under the conditions used was ascertained by measurement of the O2- release from the same cell batches. Superoxide production known as protein kinase C-dependent in Kupffer cells (Dieter et al. (1986) Eur. J. Biochem. 86, 451-457) was suppressed in a dose-dependent manner by staurosporin or after prolonged pretreatment with the phorbol ester. H-7 decreased superoxide production only slightly in high doses that severely harm the Kupffer cells. Prostaglandin E2 release, although clearly protein-kinase C-dependent in phagocytosing rat Kupffer cells, is not decreased following exposure to lipopolysaccharide in the presence of protein kinase C inhibitors.     

Effect of the 5-lipoxygenase inhibitor piriprost on superoxide production by human neutrophils

The effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1 (Piriprost) on the oxidative response was studied in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate, 13-acetate (PMA) or opsonized zymosan. Piriprost inhibited the stimulatory effect of fMLP on superoxide anion (O2-) generation, at concentrations higher than those which depress leukotriene B4 (LTB4) formation. This inhibition was overcome by increasing the concentration of fMLP. Neither exogenous LTB4 nor indomethacin were able to reverse the inhibitory effect of piriprost on fMLP action. In contrast, piriprost did not inhibit the stimulation of O2- production induced by PMA or zymosan. Piriprost behaves thus as a specific and apparently competitive antagonist of fMLP: this action does not seem to involve lipoxygenase inhibition and might be exerted at the level of the fMLP receptor or its associated mechanisms of transduction.     

Surfactant phospholipid DPPC downregulates monocyte respiratory burst via modulation of PKC

Pulmonary surfactant phospholipids have been shown previously to regulate inflammatory functions of human monocytes. This study was undertaken to delineate the mechanisms by which pulmonary surfactant modulates the respiratory burst in a human monocytic cell line, MonoMac-6 (MM6). Preincubation of MM6 cells with the surfactant preparations Survanta, Curosurf, or Exosurf Neonatal inhibited the oxidative response to either lipopolysaccharide (LPS) and zymosan or phorbol 12-myristate 13-acetate (PMA) by up to 50% (P < 0.01). Preincubation of MM6 cells and human peripheral blood monocytes with dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid component of surfactant, inhibited the oxidative response to zymosan. DPPC did not directly affect the activity of the NADPH oxidase in a MM6 reconstituted cell system, suggesting that DPPC does not affect the assembly of the individual components of this enzyme into a functional unit. The effects of DPPC were evaluated on both LPS/zymosan and PMA activation of protein kinase C (PKC), a ubiquitous intracellular kinase, in MM6 cells. We found that DPPC significantly inhibited the activity of PKC in stimulated cells by 70% (P < 0.01). Western blotting experiments demonstrated that DPPC was able to attenuate the activation of the PKCdelta isoform but not PKCalpha. These results suggest that DPPC, the major component of pulmonary surfactant, plays a role in modulating leukocyte inflammatory responses in the lung via downregulation of PKC, a mechanism that may involve the PKCdelta isoform.     

Hepatitis B virus differentially suppresses myelopoiesis and displays tropism for immature hematopoietic cells.

The hematopoietic cell lines HL-60 and THP-1 were challenged with hepatitis B virus (HBV) in vitro to study interactions between the virus and host cell. Exposure to HBV suppressed the ability of HL-60 cells to differentiate into granulocytes after treatment with retinoic acid (RA) or dimethyl sulfoxide (DMSO), and RA-induced activation of the monocytic cell line THP-1 was also suppressed. Terminal differentiation of both cell lines by phorbol 12-myristate 13-acetate (PMA) was not affected by HBV. The suppressive effect on RA- or DMSO-induced differentiation was unique to HBV, since cell exposure to human cytomegalovirus, another virus that inhibits hematopoiesis, failed to block cellular differentiation. At 5 days postinfection, extracellular viral DNA was detected in immature but not in differentiated cultures and higher levels of core antigen (HBcAg) and surface antigen (HBsAg) were seen in undifferentiated cells than in RA- or PMA-treated cells. In addition, release of HBsAg into the medium was 2 to 12 times greater in untreated cultures than for RA- or PMA-treated cells. Thus, HBV suppresses hematopoiesis by blocking the maturational development of progenitors and selectively infects immature myeloid cells compared with mature end-stage cells.     

Characterization of the amnion-derived AV3 cell line for use as a model for investigation of prostaglandin-cytokine interactions in human amnion

Increased production of prostaglandins and cytokines by amnion, particularly prostaglandin (PG) E2, interleukin (IL)-6 and IL-8, is thought to be an important event in infection-associated preterm labour. We characterized the amnion-derived AV3 cell line to determine its appropriateness as a model for investigation of the regulation of amnion cytokine and PG production. Amnion-derived AV3 cells were treated with tumour necrosis factor-alpha (TNF-alpha, interleukin-1beta (IL-1beta), epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) and IL-6, IL-8 and prostaglandin production was determined by immunoassay. Production of IL-6 and IL-8 rose dramatically with all treatments. PGE2, but not PGF2alpha or 6-keto-PGF1alpha, biosynthesis was also increased in a concentration-dependent manner with all treatments. A rapid increase in PGHS-2 (but not PGHS-1) mRNA expression was observed in response to TNF-alpha and IL-1beta. We conclude that the AV3 cell line inflammatory response profile is similar to those observed in primary amnion and other amnion-derived cell lines, and is an appropriate model for human amnion.     

Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme

The extracellular domains of many proteins, including growth factors, cytokines, receptors, and adhesion molecules, are proteolytically released from cells, a process termed "shedding." Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease-disintegrin that sheds tumor necrosis factor-alpha and other proteins. To study the regulation of TACE-mediated shedding, we examined the effects of stimulation of cells on TACE localization and expression. Immunofluorescence microscopy revealed a punctate distribution of TACE on the surface of untreated cells, and stimulation of monocytic cells with lipopolysaccharide did not affect TACE staining. Phorbol 12-myristate 13-acetate (PMA), a potent inducer of shedding, decreased cell-surface staining for TACE. Surface biotinylation experiments confirmed and extended this observation; PMA decreased the half-life of surface-biotinylated TACE without increasing the turnover of total cell-surface proteins. Soluble fragments of TACE were not detected in the medium of cells that had down-regulated TACE, and TACE was not down-regulated when endocytosis was inhibited. Antibody uptake experiments suggested that cell-surface TACE was internalized in response to PMA. Surprisingly, a metalloprotease inhibitor prevented the PMA-induced turnover of TACE. Thus, PMA activates shedding and causes the down-regulation of a major "sheddase," suggesting that induced shedding may be regulated by a mechanism that decreases the amount of active TACE on the cell surface.     

Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood

BACKGROUND: Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring. METHODS: With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population. RESULTS: Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation. CONCLUSIONS: This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.     

Pertussis toxin and H-7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide-primed macrophages. Eicosanoid release from lipopolysaccharide-primed macrophages

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1-2 micrograms/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 microM) or with phorbol 12-myristate 13-acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12-myristate 13-acetate. The presence of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12-myristate 13-acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis-toxin-sensitive, guanine-nucleotide-binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide-activated macrophages in response to A23187 and phorbol 12-myristate 13-acetate.     

Transactivation of human immunodeficiency virus type 1 long terminal repeats by cell surface tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF-alpha) is expressed in secreted and cell surface (csTNF-alpha) forms by activated monocytic and T cells. In this report, we demonstrate that csTNF-alpha may predominantly regulate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activation in the promonocytic cell line U937 and in the Epstein-Barr virus-transformed B-cell line BH1. Anti-TNF-alpha antibody suppressed both the constitutive expression of the HIV-1 LTR in BH1 cells and the expression induced by phorbol 12-myristate 13-acetate in U937 cells. This suppression was found to be mediated via csTNF-alpha. No correlation between the HIV-1 LTR activation and the secretion of TNF-alpha was evident in these cell lines. Suppression of TNF-alpha secretion by cyclosporin A or by a serine protease inhibitor did not suppress the HIV-1 LTR activation. These observations suggest a novel biological role for csTNF-alpha in the immunopathogenesis of AIDS.     

Spin-Trapping and Chemiluminescence Studies of Neutrophils from a Holstein-Friesian Calf with Bovine Leukocyte Adhesion Deficiency

The ability of neutrophils from a Holstein-Friesian calf with bovine leukocyte adhesion deficiency (the proband with a genetic deficiency of the Mac-1 (CD11b/CD18) glycoprotein corresponding to the receptor of complement iC3b) to generate oxygen radicals was examined using electron spin resonance spectrometry (ESR) combined with a spin-trapping technique and luminol-dependent chemiluminescence spectrometry. When the neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), an ESR spectrum confirming the generation of superoxide anions (O₂^-) was clearly observed in both healthy and diseased calves. However, when the neutrophils were stimulated by opsonized zymosan, appearance of the ESR spectrum was recognized in the healthy calves but not in the diseased calf. Similar results were obtained from chemiluminescence experiments.