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PubMed1979

Induction of lysosomal enzyme secretion by alveolar macrophages in response to the purified complement fragments C5a and C5a des-arg.

Purified C5a and its "inactive" form, C5a des-arg, were shown to induce secretion of acid hydrolases from rabbit alveolar macrophages (AM) in a concentration-dependent manner. Secretion increased with time to 5 times above controls by 72 hr. Concentrations of these enzymes in the cell lysates did not decrease during the incubation, suggesting that synthesis of new enzyme was occurring. The lysosomal enzyme secretion was accompanied by increased pinocytosis and release of proteolytic enzymes from the macrophages. At no time was significant lactic dehydrogenase liberated, indicating that secretion was selective and not due to cell death. Data presented also suggest that C5a des-arg induced secretion from the macrophages of a chemotactic factor for neutrophils. It was concluded that C5a and C5a des-arg may play a role in lung injury by interactions with AM, inducing the secretion of acid hydrolases and proteolytic enzymes that can cause tissue damage, and by regulating the influx of other inflammatory cells into the interstitium and air spaces.

Anaphylatoxins↗
PubMed1991

Human neutrophil migration into skin chambers is associated with production of NAP-1/IL8 and C5a.

Respiratory burst activity initiated by the chemoattractants fMLP, rC5a and rNAP-1/IL8 was investigated in human exudated and peripheral blood neutrophils. Exudated cells were isolated after migration into a skin chamber and the respiratory burst activity was measured as chemiluminescence amplified by luminol and horseradish peroxidase. The response to fMLP (5 x 10(-8) mol/l) was significantly enhanced (p less than 0.01) in the exudated cells but was significantly decreased after stimulation (5 x 10(-8) mol/l) with rC5a and rNAP-1/IL8 (p less than 0.05 and p less than 0.01, respectively). Analysis revealed that, in the chamber fluid, the activated complement C5a was generated during exudation (p less than 0.01). Determinations of NAP-1/IL8 showed that this substance was also produced and released into the chamber fluid (p less than 0.01). No correlation was found between the number of exudated cells and the amount of C5a or NAP-1/IL8 in the exudation fluid, thus indicating that, in vivo, the exudation process is controlled by multiple factors and not by the quantity of a single chemoattractant. The present study shows that NAP-1/IL8 and C5a are produced in humans during an aseptic inflammation, and that this occurs in parallel to the migration of neutrophils into the skin chambers. The significant desensitization of the exudated cells to NAP-1/IL8 and C5a reflects a previous exposure to these attractants. These results suggest that the novel tissue-derived cytokine NAP-1/IL8 plays a role in human neutrophil exudation in vivo.

Cell Movement↗
PubMed1991

Regulation of complement 5a receptor expression in U937 cells by phorbol ester.

Receptors for the anaphylactic portion of complement, C5a, are not initially expressed in the monoblastic U937 cell line, but appear as the cell is induced to differentiate by the synergistic actions of 1,25(OH)2D and cyclic adenosine monophosphate (cAMP). Phorbol myristate acetate (PMA), which activates the protein kinase C pathway (PKC), does not cause C5a receptor (C5aR) expression when used as a single agent. The induction of C5aR by the synergistic actions of 1,25(OH)2D and cAMP, however, can be augmented as much as 180% by the addition of PMA. C5aR arising in cells exposed to 1,25(OH)2D and 8,4-chlorophenylthio-cAMP have an affinity constant of about 0.4 nM as assessed by cold competition analysis. We show here that when phorbol augmentation of receptor number occurs, the affinity constant is increased by 3.6-fold. In an effort to ascertain whether the change in C5aR Kd involved a PKC-dependent event we examined whether 5-60 min exposure of C5aR-positive cells to PMA would change C5aR Kd. Acutely, PMA caused a downregulation of receptor binding with decreases in apparent receptor number out of proportion to changes in Kd. One hundred nanomolar PMA, which effects nearly complete translocation of PKC to the membrane, consistently caused a 70-90% decrease in C5a surface binding. This downregulation was proportional to PMA dose and exposure time. Micromolar concentrations of the microtubule depolymerizing agents colchicine and vinblastine caused a less drastic downregulation, about 50% of the maximal phorbol effect. Our data suggest that activation of the PKC system might acutely limit the macrophage's ability to respond to C5a; chronically, phorbols upregulate receptor expression, most likely through positive effects on C5aR gene expression.

Calcitriol↗
PubMed1989

[Effects of nicotine and cigarette smoke extracts on plasma level of complement C3a and C5a, thromboxane B2 and 6-keto PGF1 alpha in rabbits].

In the present investigation we measured plasma levels of complement C3a and C5a, thromboxane B2 (TxB2) and 6-keto PGF1 alpha in anesthetized rabbits by radioimmunoassay after the intravenous injection of nicotine and cigarette smoke extracts. Plasma levels of complement C3a in rabbits showed a significant increased and reached a maximum value 60 minutes after the intravenous injection of nicotine and cigarette smoke extract. Plasma levels of complement C5a in rabbits showed a significant increase and reached a maximum value 30 minutes after the intravenous injection of nicotine. Plasma levels of complement C5a in rabbits showed a significant increase and reached a maximum value 60 minutes after the intravenous injection of cigarette smoke extract. Plasma levels of TxB2 in rabbits showed a significant increase after the intravenous injection of nicotine and cigarette smoke extract. Plasma levels of 6-keto PGF1 alpha in rabbits did not show any change after the intravenous injection of nicotine and cigarette smoke extracts. The above results may suggest that the intravenous injection of nicotine and cigarette smoke extracts induces an increase of plasma levels of complement C3a and C5a and TxB2 in anesthetized rabbits.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Model structure for the inflammatory protein C5a.

The complement cleavage product C5a is a potent stimulant of inflammatory processes; thus, inhibition of C5a activity is of therapeutic interest. The three-dimensional structure of the major portion of C5a was modeled from the homologous C3a crystal structure by comparative modeling techniques. The model shows that core residues of C5a are completely conserved, while external residues differ from C3a. Even though the amino-terminal 12 residues of C3a are disordered in the crystal, this sequence in C5a may form an amphipathic helix. The distribution of species sequence differences in the complete C5a structure suggests a possible receptor binding site.

Amino Acid Sequence↗
PubMed1984

Characterization of the anaphylatoxin inactivator and chemotactic factor inactivator activities during cardiopulmonary bypass.

Complement activation productive of phlogistic products has been suggested as one of the major mechanisms of the pump lung syndrome associated with cardiopulmonary bypass (CPB) surgery. Recent studies have demonstrated the presence of circulating C3a antigens in the serum of patients undergoing CPB and have suggested that the vasoactive nature of C3a may contribute directly to the interstitial edema and vascular changes seen in pump lung syndrome. In an effort to unravel the underlying mechanisms of pump lung syndrome, we undertook investigations to determine whether CPB and associated complement activation would alter the serum levels of the major regulators of both C3a and C5a complement split products. These serum regulators designated chemotactic factor inactivator (CFI) and anaphylatoxin inactivator (AI) were measured in the serum of patients undergoing CPB. In these studies, we demonstrated that during CPB a rapid and dramatic drop in the anaphylatoxin inactivator activities occurred within the first 10 minutes of CPB. These lowered AI levels were maintained throughout the CPB but AI levels returned to normal within 24 hours postsurgery. CFI levels were generally maintained throughout the CPB surgery with only minimal depressions in CFI levels during or after CPB surgery. These studies clearly demonstrate that the major regulator system of the complement-derived vasopermeability factors (C3a and C5a) is dramatically depressed during cardiopulmonary bypass and may suggest that the mechanisms of interstitial edema associated with pump lung syndrome may at least, in part, be related to the loss of the serum regulator enzyme carboxypeptidase N, also designated AI.

Anaphylatoxins↗
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↗
PubMed1984

Various authentic chemoattractants mediating leukocyte adherence inhibition.

This study resulted from the finding that cancer extract-induced leukocyte adherence inhibition (LAI) depends on release of arachidonic acid metabolites of which leukotriene B4 (LTB4) is a chemoattractant. Leukotrienes, the chemotactic fragment of the fifth component of complement (C5a des arg), N-formyl-L-methionylyl-L-leucyl-L-phenylalanine (FMLP), platelet-activating factor (PAF), and phorbol myristate acetate (PMA) increased the nonadherence of human leukocytes to glass with bell-shaped dose-response curves. For maximum nonadherence, the optimum concentration was about 2 X 10(-11) M LTB4, 10(-9) M C5a des arg, 2 X 10(-8) M FMLP, 2 X 10(-9) M PAF, and 2 X 10(-8) M PMA. Of the adherent leukocytes, 29% became nonadherent. Higher concentrations of leukotriene E4, FMLP, PAF, and PMA induced leukocyte hyperadhesiveness. Chemoattractant-induced LAI was antagonized by inhibitors of glycolysis, oxidative metabolism, electron transport, microfilaments, and microtubules, whereas the same concentration of inhibitors did not alter the adherence of leukocytes to glass in control tubes. T-cells, neutrophils, and mononuclear cells showed LAI to LTB4, C5a des arg, and FMLP. Moreover, no additive effect was observed when leukocytes exposed to a first chemoattractant at its optimum concentration were exposed to a second. A competitive inhibitor of leukotriene slow-reacting substance of anaphylaxis, FPL 55712, blocked in a dose-response fashion LTB4-, C5a des arg-, and FMLP-triggered LAI. Eicosatetraenoic acid and nordihydroguaiaretic acid, cyclo-oxygenase-lipoxygenase pathway inhibitors, also markedly antagonized LTB4-, C5a des arg-, and FMLP-triggered LAI. Indomethacin (10(-5) M), piroxicam (10(-6) M), and aspirin, cyclooxygenase antagonists, negated LTB4-, C5a des arg-, and FMLP-induced LAI. Prostaglandins E2, E1, and F2 and prostacyclin did not trigger LAI. However, selective thromboxane synthetase antagonists completely negated chemoattractant-triggered LAI. Our studies indicated that chemoattractants stimulated leukocytes to produce thromboxanes, which increased leukocyte nonadherence.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
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↗
PubMed1980

Stimulus-specific deactivation of chemotactic factor-induced cyclic AMP response and superoxide generation by human neutrophils.

The responses of isolated human peripheral neutrophils to either simultaneous or sequential additions of two chemotactic factors were studied. Simultaneous additions of formyl-methionyl-leucyl-phenylalanine (10-100 nM) and the fifth component of complement, C5a (1-10 microliters/ml), evoked partially additive responses of membrane depolarization as measured by the fluorescent dye 3,3'-dipropyl-thiocarbocyanine, a transient elevation of intracellular cyclic AMP (cAMP), and superoxide (O2-) generation as assessed by ferricytochrome c reduction. Preincubation of the cells with either formyl-methionyl-leucyl-phenylalanine or C5a alone caused dose-dependent inhibition of the depolarization, the cAMP increase, and O2- release induced by a subsequent exposure to an optimal dose of the same stimulus, i.e., deactivation occurred. In contrast, when cells were treated with one chemotactic factor and then exposed to the other stimulus, the cells exhibited a normal response of peak depolarization, the rise in cAMP, and O2-0 production i.e., cross-deactivation failed to occur. The results imply that deactivation of these phenomena is stimulus specific. Further, these observations are consistent with the hypothesis that cross-deactivation of chemotaxis is mediated by one or more processes that are irrelevant to O2- generation, and that occur distal to the depolarization and cAMP steps in the sequence of neutrophil activation: possibly microtubule polymerization and orientation.

Anaphylatoxins↗
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↗