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PubMed1988

Endotoxemia and neutrophil activation in vivo.

There is a growing body of data to suggest that marginated granulocytes mediate much of the pulmonary damage observed during endotoxemia. The mechanism(s) by which endotoxemia initiates neutrophil margination and cytotoxicity remain either controversial or unknown. The objectives of this study were 1) to determine the temporal relationship between endotoxin-induced decreases in mean arterial pressure and circulating neutrophils, 2) to monitor neutrophil activation in vivo by measuring myeloperoxidase (MPO) activity in the plasma and lymph, and 3) to assess the interaction between endotoxin and complement in activation of neutrophilic oxidative metabolism in vitro. We found that a bolus injection of endotoxin causes a concurrent decrease in both mean arterial pressure and circulating neutrophils at 2 min postinfusion. Blood pressure recovered to approximately 70% of control values by 180 min, whereas circulating neutrophils remain depressed at 20% of control values for the entire experimental period. Using MPO as a marker for neutrophil activation, we found that infusion of endotoxin produces a dramatic increase in plasma and lymph MPO activity, suggesting activation of neutrophilic metabolism in vivo. In vitro data showed that both endotoxin and plasma were required for optimal neutrophilic degranulation and superoxide formation. We conclude that 1) the appearance of MPO in the plasma (or lymph) may be a useful neutrophil marker for neutrophil activation in vivo and may prove useful in following the course of neutrophil-mediated tissue injury during endotoxemia, and 2) endotoxin-activated complement (C5a) activates neutrophils to produce cytotoxic oxidants.

Animals↗
PubMed1991

Differential inhibition and potentiation by cell-permeant analogues of cyclic AMP and cyclic GMP and NO-containing compounds of exocytosis in human neutrophils.

The chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), complement C5a and platelet-activating factor (PAF), induce beta-glucuronidase release and aggregation and an increase in cytosolic Ca2+ [Ca2+]i in human neutrophils. We studied the roles of cAMP and cGMP in neutrophil avtivation, using their cell-permeant analogues, N6,2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) and N2,2'-O-dibutyryl guanosine 3':5'-cyclic monophosphate (Bt2cGMP) and the NO-containing compounds, sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and its prodrug, molsidomine (SIN-10). Bt2cAMP, Bt2cGMP, SIN-1 and SIN-10 but not SNP inhibited exocytosis induced by fMet-Leu-Phe. Superoxide dismutase potentiated the inhibitory effect of SIN-1. Bt2cGMP and SNP potentiated C5a-induced beta-glucuronidase release, Bt2cAMP, KCN, SIN-1 and SIN-10 being ineffective. KCN partially reversed the stimulatory effect of SNP, and in the presence of superoxide dismutase, SIN-1 potentiated C5a-induced exocytosis. PAF-induced beta-glucuronidase release was not affected by Bt2cAMP, Bt2cGMP, SNP and SIN-1. Bt2cGMP was more effective than Bt2cAMP to inhibit aggregation and the increase in [Ca2+]i induced by fMet-Leu-Phe at submaximally effective concentrations. C5a-induced rises in [Ca2+]i were not affected by Bt2cAMP and Bt2cGMP. Bt2cAMP but not Bt2cGMP inhibited the effect of PAF at submaximally effective concentrations on [Ca2+]i. Our data suggest (I) that Bt2cGMP and Bt2cAMP differentially modulate neutrophil activation, that (II) NO-containing compounds partially mimic the effects of Bt2cGMP on exocytosis and that (III) cGMP plays an inhibitory role in fMet-Leu-Phe- and a stimulatory role in C5a-induced beta-glucuronidase release.

1-Methyl-3-isobutylxanthine↗
PubMed1989

[Experimental research on the pathogenic mechanisms in adult respiratory distress syndrome].

In order to study the pathogenic mechanisms of adult respiratory distress syndrome (ARDS), the neutrophil aggregation activity, platelet aggregation rate and levels of thromboxane, prostacyclin, superoxide dismutase, lipoperoxides, neutrophil elastase, alpha 1-antitrypsin and angiotensin conversion enzyme were measured in 84 experimental dogs. Under the action of activated complement C5a, polymorphonuclear leukocyte aggregation with increased release of oxygen radicals play an important role in the pathogenesis of ARDS.

Animals↗
PubMed1990

Effects of leukocyte activation on myocardial vascular resistance.

The contribution of the leukocyte, particularly the granulocyte, to the tissue injury resulting from the inflammatory response accompanying organ ischemia is a subject of intense, current interest. Leukocytes are large and viscous cells which adhere to vascular endothelium, and are a source of a variety of toxic and vasoactive substances. There are several lines of evidence indicating their involvement in the development of abnormal and heterogeneous tissue perfusion in a wide variety of pathologic states. They have been implicated in the capillary stasis and no-reflow following hemorrhagic shock, and in ischemia and reperfusion of skeletal muscle, brain, and heart. The mechanisms responsible for the detrimental influence of the granulocyte on tissue perfusion include their inherent rheologic properties, their role in the generation of vascular smooth muscle-constricting substances, and their potential for damaging vascular endothelium. One contributing aspect of the inflammatory response is leukocyte activation by products of the complement cascade. In our in vivo model system, stimulation of the granulocyte with activated complement C5a (intracoronary) is associated with myocardial ischemia and a transient myocardial accumulation of granulocytes. The enhanced generation of thromboxane A2 and leukotrienes appears to be primarily responsible for this increase in coronary vascular resistance.

Animals↗
PubMed1992

Purification of human blood basophils using negative selection by flow cytometry.

Basophils were purified from peripheral blood of normal donors using Percoll discontinuous gradients and negative selection by flow cytometry. The mean purity of basophils obtained was 84.7 +/- 4.1 (s.d.)% (range 77.3-90.0%, n = 13). The overall yield of these procedures was 16.0 +/- 2.6% (range 11.0-19.9%, n = 13), and cell viability of purified basophils exceeded 90%. Properties of highly purified basophils obtained by flow cytometry did not differ from those of partially enriched basophil preparations from Percoll discontinuous gradients in respect of: (i) intracellular histamine content; (ii) percentage of spontaneous histamine release in buffer; and (iii) percentage of histamine release triggered by ionophore A 23187 or anti-IgE. Moreover, purified basophils responded chemotactically to complement C5a in a dose-dependent manner. These findings suggest that our procedure for purification of human basophils does not affect the functions of basophils and may be useful for in vitro studies on the role of basophils in hypersensitivity reactions such as bronchial asthma.

Basophils↗
PubMed1992

Effects of cytokines on human basophil chemotaxis.

Basophil chemotactic activity (BCA) of eight recombinant human (rh) cytokines was examined. Highly purified basophils were obtained by Percoll discontinuous gradients, followed by negative selection using flow cytometry. Then BCA was measured by means of modified Boyden chamber method. Both interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) had much more potent BCA than complement C5a, leukotriene B4 and platelet activating factor, well known as granulocyte chemotactic factors. Chemotaxis rather than chemokinesis was shown in chequerboard analysis of basophil migration induced by IL-3 and GM-CSF. Relatively high concentrations of IL-5 also induced basophil migration, although predominantly chemokinetic. IL-8 had apparent BCA, which was not so high as that of C5a. In contrast, IL-2, IL-4, interferon(IFN)-gamma and granulocyte colony-stimulating factor (G-CSF) had no significant BCA. These findings suggest that IL-3, IL-5, GM-CSF and, perhaps, IL-8 have an effect on basophil migration as well as modulation of basophil mediator release and may provide some insight into the basophil accumulation observed in late-phase allergic responses.

Basophils↗
PubMed1985

Chemotactic responses of human peripheral blood monocytes to the complement-derived peptides C5a and C5a des Arg.

We examined responses of human peripheral blood polymorphonuclear leukocytes (PMN) and monocytes to the highly purified human complement-derived peptides C5a and C5a des Arg. As reported previously, C5a proved to be approximately 10- to 20-fold more potent than C5a des Arg as a chemoattractant for human PMN. C5a also was more potent than C5a des Arg in causing PMN to acquire a polarized morphology. In contrast, we found that human monocytes do not distinguish between C5a and C5a des Arg when these peptides are used as chemoattractants. In two different assay systems, both peptides acted at identical concentrations to stimulate suboptimal and optimal migration of monocytes. Human monocytes also did not distinguish between C5a and C5a des Arg when these peptides were used as inducers of polarization. Studies performed with functionally active, [125I]-labeled C5a and C5a des Arg, however, demonstrated that binding of C5a des Arg to monocytes differed from binding of C5a. Although [125I]-C5a des Arg appeared to bind to the same receptor as [125I]-C5a, binding of labeled C5a des Arg occurred with an affinity that was approximately 100-fold less than that observed with labeled C5a. These results indicate that leukocyte chemotactic and polarization responses to C5a and C5a des Arg vary, depending on the target cell type.

Adult↗
PubMed1992

Human T lymphocytes and T-cell lines as target cells for lymphocyte chemotaxis.

We have observed that freshly isolated T lymphocytes from healthy donors give a chemotactic response to complement C5a in 26 of 55 individuals and to epidermal lymphocyte chemotactic factor in 15 of 23 donors using 51chromium-labelled lymphocytes in a double-filter Boyden chamber system. The reason for a lack of demonstrable chemotaxis among some cell populations is unknown, but it makes donor selection important when studying lymphocyte chemotaxis. In order to obtain a standardized screening assay for T lymphocyte chemotactic activity, we investigated a number of T-cell lines or T-cell-related cell lines such as HuT78, Jurkat, MOLT4, K562 and 1301. We observed that HuT78, K562 and Jurkat showed chemotactic responses to a variety of mediators, whereas 1301 showed chemotaxis only towards C5a, and MOLT4 was completely negative. The HuT78 cell line, which is derived from a patient with Sézary's syndrome, exhibited the highest chemotactic capacity similar to freshly isolated T lymphocytes. The only difference was its chemotactic response towards stimulation with recombinant interleukin-1 alpha and beta, which did not induce chemotaxis in human peripheral blood T lymphocytes in the Boyden chamber assay. We conclude that HuT78 can be used in screening various inflammatory mediators for their potential T lymphocyte chemotactic properties.

Biological Assay↗
PubMed1986

Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B.

Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B beta-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at approximately 10(-8) M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionyl-leucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10(-7) M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and beta-glucuronidase), or the production of superoxide anion. These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover. The capacity of hFpB to cause PMN chemotaxis without causing concurrent release of lysosomal enzymes or the production of superoxide anion is further evidence for the complexity of PMN responses to chemotactic agents.

Actins↗
PubMed1991

Mechanisms of contraction induced by human leukocytes in normal and atherosclerotic arteries.

Activation of leukocytes results in the release of a variety of vasoactive substances that may modulate vascular tone. We studied the effect of human polymorphonuclear (PMN) and mononuclear (MONO) leukocytes on quiescent femoral arteries in vitro. Arteries were obtained from normal and atherosclerotic cynomolgus monkeys. In normal arteries, stimulation of PMNs (3 and 5 x 10(6) cells/ml) with either thrombin (5 units/ml) or complement C5a (0.5 micrograms/ml) resulted in endothelium-independent contraction (approximately 25% of maximum contraction with 80 mM KCl). Vasocontraction was augmented in the presence of superoxide dismutase (150 units/ml) and was significantly impaired in the presence of the hydroxyl radical scavengers mannitol (20 mM) and deferoxamine (1 mM). Catalase (1,200 units/ml) or L-alanine (20 mM) did not modify this effect of PMNs. In contrast to PMNs, vasocontraction in response to MONOs was not altered by the addition of radical scavengers. Pretreatment of PMNs and MONOs with indomethacin (10 microM) or nordihydroguaiaretic acid (20 microM) did not influence vascular responses. Supernatant of thrombin-stimulated PMNs and MONOs also produced vasocontraction (approximately two thirds of the effect of intact cells). This vasocontractor factor (or factors) was heat stable (30 minutes, 95 degrees C) and had a molecular weight less than 1,000 as determined by ultrafiltration. Stimulation of MONOs or PMNs (3 and 5 x 10(6) cells/ml) produced a similar response in normal arteries. In contrast, the constrictor response in atherosclerotic arteries to MONOs (5 x 10(6) cells/ml) was significantly greater than to PMNs. We conclude that stimulated human PMNs and MONOs contract arteries in vitro by release of at least two factors. One factor appears to be heat stable, with a molecular weight less than 1,000. The vascular response to PMNs, but not to MONOs, appears to involve the generation of hydroxyl radicals. The response to MONOs is greater than the response to PMNs in atherosclerotic, but not in normal, arteries.

Animals↗
PubMed1985

Elevated C3 anaphylatoxin levels in synovial fluids from patients with rheumatoid arthritis.

Because cleavage products of the third component of complement augment inflammation and may contribute to arthritis, we used a competitive inhibition radioimmunoassay to measure levels of the low molecular weight cleavage products of the third component of complement, C3a and C3adesArg, in 72 synovial fluid samples. Mean levels of C3a/C3adesArg were more than sevenfold higher in 41 patients who had rheumatoid arthritis than in 15 patients who had degenerative joint disease or 5 patients who had traumatic arthritis. Striking elevations were also present in 2 patients who had acute gouty arthritis. A calculation of the fraction of intraarticular C3 cleaved showed that the patients with rheumatoid arthritis had a mean C3 cleavage of 11.6 +/- 11.0%, which was significantly higher than values of less than 1.5% for patients with degenerative joint disease or traumatic arthritis. In rheumatoid arthritis and gouty arthritis, specific immunoassay identified substantial quantities of the initial C3 cleavage fragments.

Arthritis, Rheumatoid↗
PubMed1990

Characterization of human phagocytic cell receptors for C5A and platelet activating factor expressed in Xenopus oocytes.

Platelet activating factor (PAF) and the active cleavage product of the fifth component of complement, C5a, are potent anaphylotoxins and mediators of inflammation. Both substances engage distinct guanine nucleotide binding regulatory protein-coupled receptors on a variety of cell types, thereby activating a signaling cascade that results in the mobilization of intracellular calcium stores, and in functional responses such as neutrophil chemotaxis and smooth muscle contraction. Little is known about the structure of PAF and C5a receptors or about the intracellular signaling pathways used by them. We have used the Xenopus oocyte expression system to demonstrate acquired C5a and PAF receptor activity in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 differentiated with dibutyryl cAMP. Activity was determined by measuring acquired ligand-dependent efflux of intracellular 45Ca2+ and by measuring ligand-activated transmembrane currents in voltage clamped oocytes. C5a receptor activity was confined to a single sharp peak in sucrose gradient fractionated RNA that corresponded to a transcript size of 2 kb. In contrast, PAF receptor activity was broadly distributed in size fractionated RNA from 3.5 to 6 kb. This suggests that multiple transcripts of different sizes may encode a functional PAF receptor. Both ligands activated their respective receptor in a concentration-dependent and a structure-dependent manner. The acquired C5a-dependent calcium efflux activity was inhibited by pertussis toxin whereas the PAF receptor activity was not, suggesting that the two receptors couple to different G-proteins. These data establish the Xenopus oocyte as a model system for studying the molecular and functional properties of the C5a receptor and the PAF receptor(s) of phagocytic cells.

Animals↗
PubMed1988

Expression of C5a anaphylatoxin receptor in monoblastic cells involves facilitation of an adenosine 3',5'-monophosphate-dependent process.

We have previously reported a synergistic effect of 1,25-dihydroxyvitamin D [1,25-(OH)2D] and agents that elevate intracellular cAMP to induce the expression of the C5a complement receptor in U937 cells. In this report we examine the mechanism of this synergy, considering the hypothesis that the steroid hormone works by facilitating what is ultimately a cAMP-dependent process. We show that U937 cells cultured with 1,25-(OH)2D alone before culture with prostaglandin (PGE2) alone will express C5a receptor (an average of 55 +/- 4% of the receptors expressed with continuous exposure of cells to both agents; P less than 0.05). The reverse, PGE2 followed by 1,25-(OH)2D, causes very little receptor induction. This demonstrates the ability of 1,25-(OH)2D to induce changes in the state of the cell, such that activation of cAMP-dependent protein kinase has effects that are otherwise not seen, in other words 1,25-(OH)2D can prime the cell for the subsequent action of the cAMP messenger system. Furthermore, we are able to substitute, during the priming period, the protein synthesis inhibitor cycloheximide (CHX) for 1,25-(OH)2D. Cells cultured for 24 h with CHX will express C5a receptor when cultured for a second 2-day period with PGE2 at about 77 +/- 7% of the amount obtained with simultaneous exposure to 1,25-(OH)2D and PGE2. The CHX effect is time dependent and visible after 2 h. CHX is not synergistic with 1,25-(OH)2D. Other agents that can also substitute for 1,25-(OH)2D, but not for cAMP, in facilitating C5a receptor expression include retinoic acid and ionomycin, but with less potency. The 1,25-(OH)2D and PGE2 synergy is sensitive to the presence of isobutylmethylxanthine, implicating its dependence on the maintained elevation of intracellular cAMP levels. The synergy does not appear to be sensitive to changes in extracellular or intracellular calcium. We conclude from these results that 1,25-(OH)2D may promote the expression of C5a receptor in these cells in a fashion similar to that by which CHX potentiates other genes, i.e. that 1,25-(OH)2D increases levels of the mRNA encoding the C5a receptor. The mechanism of cAMP's subsequent and necessary action in the induction of C5a receptor expression is not yet clear.

1-Methyl-3-isobutylxanthine↗
PubMedDate not supplied

Effects of cigarette smoking on blood levels of leukotrienes and plasma levels of complements C3a and C5a in healthy volunteers.

We reported previously that cigarette smoking induces bronchoconstriction. In the present investigation we measured the level of peripheral venous blood leukotrienes, which are known to cause marked bronchoconstriction, and plasma complement C3a and C5a, which are known to have chemotactic and anaphylatoxic activities. All of these showed a significant increase after cigarette smoking. These data may suggest that the activation of complement induces the release of leukotrienes, and that these leukotrienes cause bronchoconstriction. It appears likely, therefore, that cigarette smoking induces an increase in peripheral venous blood leukotrienes and complement C3a and C5a, and that such an increase may elicit bronchoconstriction.

Adult↗
PubMed1975

Quantification of mouse macrophage chemotaxis in vitro: role of C5 for the production of chemotactic activity.

Delineation of the mechanisms of macrophage accumulation at local tissue sites will further our understanding of immunologically mediated host resistance to infectious and neoplastic diseases. Since mice are frequently used for the study of immune function, we developed a method for the quantification of mouse macrophage chemotaxis in vitro. By this method it was found that the fifth component of complement is necessary for the production of chemotactic activity in mouse serum by inflammatory agents such as endotoxin or aggregated gamma globulin. The majority of macrophage chemotactic activity produced by these agents in mouse serum can be attributed to a low-molecular-weight (ca. 15,000) chemotactic factor. The data suggest that this factor is the biologically active cleavage product of the fifth component of complement, C5a.

Animals↗
PubMed2026

Qingfei Dayuan granules alleviate the inflammatory response in lipopolysaccharide-induced acute lung injury mice by inhibiting the Nf-κB signaling pathway and regulating the complement pathway.

OBJECTIVES: The study aimed to explore the effects and mechanisms by which Qingfei Dayuan granules (QFDY) mitigate pulmonary inflammation in lipopolysaccharide (LPS)-induced acute lung injury (ALI). METHODS: We established an ALI mouse model by intraperitoneal injection of LPS. HE, Transmission electron microscopy, ELISA assay of inflammatory cytokines, and immunohistochemistry (IHC) were used to assess the degree of lung injury and inflammation. Utilizing network analysis and proteomics analysis, the potential targets and pathways of QFDY were identified. Western blot, IHC, and qRT-PCR analysis were used to evaluate the potential mechanism of QFDY. Additionally, the chemical composition of QFDY were performed using UPLC-MS/MS. KEY FINDINGS: QFDY reduced the pathologic changes and inflammatory cell infiltration in lung tissue inflammation. Network and proteomic analysis showed that the mechanism of QFDY protection against ALI is closely related to the Nuclear factor-kappa B (NF-κB) signaling pathway and complement pathway. Animal experiments showed that Qingfei Dayuan granules (QFDY) significantly reduced the levels of IL-1β, IL-6, TNF-α, and lung tissue F4/80-positive alveolar macrophages. Additionally, western blot and qRT-PCR analyses showed the inhibition of the NF-κB pathway. Notably, the levels of mannose-binding lectin (MBL2) were significantly increased, while complement C3a and complement C5a proteins were reduced in the QFDY group compared to the LPS group. CONCLUSIONS: QFDY suppressed the inflammation in LPS-induced ALI by inhibiting the NF-κB and complement pathway.

Animals↗