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PubMed1986

Cellular receptors to the anaphylatoxins C3a and C5a.

A single component in the plasma membrane of human polymorphonuclear leukocytes (hPMN) has been identified as the binding site for C5a. Labelled human C5a can be cross-linked to a 48,000-Mr membrane component on the hPMN surface by using the bifunctional reagent ethylene glycol bis(succinimidyl succinate). The membrane component is believed to be the C5a receptor or the binding subunit of a C5a receptor complex. Our ligand-uptake data indicate that the hPMN has high-affinity binding sites for C5a with a Kd of the order of 1-2 nM and an estimated 50,000-113,000 binding sites/cell. Preliminary binding studies of C3a and C5a to rat peritoneal mast cells indicate that non-specific uptake by these cells is so great that it obscures characterization of specific receptor interactions. Data recently reported [Gervasoni, Conrad, Hugli, Schwartz & Ruddy (1986) J. Immunol. 136, 285-292] suggest that non-specific binding of C3a to mast cells is caused by electrostatic interactions between the cationic ligand and anionic heparin-proteoglycan on the cell surface, with an additional complication of the bound ligand undergoing proteolytic degradation. It is therefore proposed that synthetic analogue peptides designed to minimize non-specific interactions with the cell will be useful tools for demonstrating anaphylatoxin receptors on mast cells and may prove essential for receptor isolation.

Animals↗
PubMed1987

The phenomenon of leukergy: induction and detection of leukocyte aggregation in whole human blood.

Leukocyte aggregation is involved in the generation of vascular damage during various inflammatory conditions. So far, in vitro leukocyte aggregation has been studied by mixing patients' plasma or serum with leukocytes of a normal donor in a platelet aggregometer. We evaluated the leukergy phenomenon, that is, the occurrence of aggregated leukocytes in peripheral blood of patients with inflammatory disorders, as an alternative tool to the former method. Leukocyte aggregation could be induced by zymosan-activated plasma, phorbol myristate acetate, and formyl-methionyl-leucyl-phenylalanine, as well as by the calcium ionophore A23187 in vitro in whole human blood. This aggregation was blocked by various lipoxygenase and cyclooxygenase pathway inhibitors. Leukergy was diagnosed in peripheral blood of patients with inflammatory disorders and was generally not associated with elevated concentrations of immune complexes, lactoferrin, C3a des Arg, or C5a des Arg. To the best of our knowledge, this is the first report on leukocyte aggregation studies performed in whole blood. Such study permits evaluation of the aggregation phenomena of leukocytes in their natural milieu. Furthermore, it is possible with this method to obtain direct visualization of leukocytic aggregates in the peripheral blood of patients. The significance of our results and their possible implications are discussed in light of the pertinent literature.

Adult↗
PubMed1987

Antigenic identification of chemotactic factor inactivator in normal human serum and bronchoalveolar lavage fluid.

Several proteins have been described that can modulate the activity of the complement component C5a, a potent chemoattractant for neutrophils. One of these inhibitors has been termed chemotactic factor inactivator (CFI). We hypothesized that CFI was antigenically present in normal human serum and that antigenic levels would correlate with the ability of serum to inhibit C5a. To test this hypothesis, CFI was purified from normal human serum, antibodies to CFI were developed in rabbits, and these reagents were used to develop an enzyme-linked immunoadsorbent assay to measure CFI. Sera from 32 normal volunteers were assayed for CFI and found to contain 77 +/- 29 micrograms/ml (range 17 to 137 micrograms/ml). Partially purified CFI from normal human sera was found to inhibit 61% +/- 9% (range 45% to 75%) of the ability of C5a to attract human neutrophils. Importantly, the ability of CFI to inhibit C5a-induced neutrophil chemotaxis correlated with the antigenic amounts of CFI (r = 0.68, P less than 0.05), suggesting that CFI is a major inhibitor of C5a. This was confirmed by the finding that (1) all C5a inhibitory activity coprecipitated with CFI by ammonium sulfate precipitation (45% to 65% saturation), and (2) depletion of this ammonium sulfate fraction of CFI resulted in a major loss in its ability to inhibit the chemotactic activity of C5a (57% vs. 8% inhibition, P less than 0.01). To determine whether CFI could play a role in the modulation of inflammation at tissue sites, normal bronchoalveolar lavage fluid was evaluated for the presence of CFI. CFI was identified in all fluids (mean 0.50 +/- 0.09 micrograms/mg albumin, range 0.14 to 1.43 micrograms/mg albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Polymorphonuclear leukocyte chemotaxis in systemic lupus erythematosus.

Studies completed thus far indicate that normal human serum contains a specific anionic polypeptide (cochemotaxin) that permits low concentrations of C5a desarg to exhibit chemotactic activity for human polymorphonuclear leukocytes. Under conditions of limited complement activation in vivo, the cochemotaxin may play an important physiologic role by amplifying the chemotactic activity of C5a desarg. The "complex" of C5a desarg plus cochemotaxin probably accounts for most of the chemotactic activity that is generated in human serum after complement activation and also appears to be the target of the heat stable cationic protein inhibitor (Bb fragment of Factor B) that is present in serum from some patients with active systemic lupus erythematosus.

Chemotaxis, Leukocyte↗
PubMed1986

A rapid method for monocyte isolation and chemotaxis.

A rapid method for isolating human peripheral blood monocytes and studying chemotaxis is described. The cells are isolated at a mean purity of 60% (+/- 10%) using Dextran. T500 sedimentation of whole blood, followed by centrifugation of the leucocyte-rich plasma obtained on a discontinuous Percoll gradient (density 1.067 g/ml). Chemotaxis was performed using a 48-well chamber and the chemoattractants activated serum (C5a) and FMLP and was proven by checkerboard analysis. Using this technique pooled serum was found to be activated by the PVP-coated polycarbonate filter in use, producing the attractant C5a. At high concentrations this produced a chemotactic response identical to zymosan-activated serum (ZAS). This technique requires small volumes of blood (15 ml) and enables monocyte isolation and chemotaxis to be completed in approximately 4 hr using leucocyte preparations in which the monocyte is the predominant cell. It therefore facilitates the sequential investigation of monocyte function in patients.

Cell Separation↗
PubMed1986

Monocyte dysfunction in severe trauma: evidence for the role of C5a in deactivation.

Consecutive severely traumatized patients (n = 16) requiring intensive care underwent serial monitoring of complement activation and monocyte migratory function with the chemoattractant activated serum (C5a) and formyl-methionyl-leucyl-phenylalanine (FMLP). Complement was found to be activated, and chemotaxis to C5a was correspondingly depressed maximally at a mean 5 to 7 days after injury (p = less than 0.01). The migratory response to FMLP was within the normal range throughout. Conversely, in a consecutive series of patients undergoing aortoiliac bypass grafting (n = 11), there was no evidence of complement activation, and monocyte migratory function remained normal for both C5a and FMLP. These data suggest that in patients with severe trauma, the activation of complement, particularly the fifth component (C5a), reduces the migratory responsiveness of circulating monocytes to C5a. This reduction in a host-response mechanism may explain the propensity to infection and poor wound healing seen in patients with severe trauma and also indicates that C5a, thought to be the major in vivo chemoattractant for leukocytes, has profound systemic actions.

Adult↗
PubMed1986

The effects of methylprednisolone on complement-mediated neutrophil activation during cardiopulmonary bypass.

Complement-mediated neutrophil activation (CMNA) has been implicated as an important pathophysiologic mechanism contributing to acute microvascular lung injury in the adult respiratory distress syndrome (ARDS). Using cardiopulmonary bypass (CPB) as a clinical model for complement-mediated microvascular injury, we studied the effects of methylprednisolone (MPSS) pretreatment on manifestations of CMNA in 28 pediatric patients undergoing CPB. Six patients not receiving MPSS served as controls. Results demonstrated that MPSS did not prevent complement activation as noted by 4.5- and 7.7-fold increases in plasma C3a des Arg levels during and immediately after CPB, respectively. However, detectable in vivo and in vitro manifestations of CMNA were altered. Neutropenia during CPB was attenuated to 65% of prebypass values compared with 47% in the control group. Neutrophil selective chemotactic desensitization toward C5a/C5a des Arg during the on bypass and postbypass periods was evident in the control group (0.41 and 0.76 cm specific migration, respectively) and prevented in the MPSS group (1.55 and 2.00 cm specific migration, respectively). We conclude that CMNA during CPB is ameliorated and/or prevented by MPSS pretreatment. These findings suggest that MPSS pretreatment may ameliorate complement-mediated microvascular (lung) injury in CPB and ARDS.

Anaphylatoxins↗
PubMed1986

The membrane attack complex in complement-mediated glomerular epithelial cell injury: formation and stability of C5b-9 and C5b-7 in rat membranous nephropathy.

Using a model of rat membranous nephropathy (MN), we examined the relationship between the development of glomerular epithelial cell injury and the formation and stability of the membrane attack complex (MAC) of complement. Isolated rat kidneys were perfused with buffered bovine albumin (BSA) or various plasmas (complement source). Kidneys containing nephritogenic amounts of complement-fixing sheep antibody to glomerular epithelial antigens (aFx1A) perfused with BSA (n = 5), and normal kidneys perfused with normal human plasma in BSA (50% v/v, n = 6) excreted 0.30 +/- 0.02 mg protein/min/g during 90 min perfusion (control groups). When normal plasma was added to the perfusate of aFx1A kidneys at concentrations of 12.5, 25, and 50% v/v, protein excretion rose in a time- and concentration-dependent manner. Perfusions with 25% plasma resulted in baseline proteinuria from 0 to 20 min that increased to 2.8 +/- 0.9 mg/min/g at 20 to 40 min and 8.6 +/- 2.1 at 40 to 60 min (n = 4, p less than 0.01 vs control groups). Removal of plasma at 20 min did not prevent this rise in protein excretion (3.9 +/- 2.4 and 5.8 +/- 2.6 mg/min/g at 30 to 40 and 55 to 65 min respectively, p less than 0.01, n = 4). Perfusion of aFx1A kidneys with C8-deficient (C8D) human plasma (25% v/v, n = 4) or C6D rabbit serum (25% v/v, n = 2) independently produced low levels of proteinuria comparable with BSA, but in combination, the two reagents restored enhanced protein excretion (n = 2). In aFx1A kidneys containing C5b-7, addition of C8 and C9 (C6D serum) after intervals of 20, 60, or 90 min immediately reconstituted heavy proteinuria. Thus, the magnitude of MAC-induced glomerular epithelial injury in rat MN is related to the complement dose. Altered glomerular permeability is delayed with respect to the onset of complement activation. Once sufficient C5b-9 is formed, proteinuria can develop despite cessation of new MAC assembly, implying that C5b-9 persists after formation. Moreover, the C5b-7 MAC intermediate is not eliminated rapidly in this model.

Animals↗
PubMed1986

Enhancement of human polymorphonuclear leukocyte adherence to plastic and endothelium by phorbol myristate acetate. Comparison with human C5a.

The adherence of circulating neutrophils to vascular endothelium represents a necessary step in the chemotactic emigration of neutrophils to extravascular inflammatory sites. Studies of neutrophil adherence induced by phorbol myristate acetate (PMA) were undertaken to determine the ability of a nonchemotactic neutrophil stimulus to provoke increased adherence. The authors found that the adherence of human neutrophils to plastic surfaces or confluent monolayers of endothelial cells is enhanced in a concentration-dependent fashion by exposure of neutrophils to PMA. The effect of PMA concentration (0.1-5.0 ng/ml) on increased neutrophil adherence parallels that observed for superoxide anion generation and release of lysosomal enzymes from specific granules. Whereas complement C5a-treated neutrophils exhibited a fourfold to fivefold increase in adherence to endothelial cells, PMA-treated neutrophils showed a 10-fold to 20-fold increase. The ability of PMA to cause increased neutrophil adherence to endothelium appeared to be directed primarily at the neutrophil. Pretreatment of neutrophils with PMA was as effective as coincubation in causing increased adherence to plastic surfaces or confluent cultured endothelial cells, but pretreatment of endothelial cells with PMA failed to promote neutrophil adherence. Alteration of neutrophil cytoskeletal structures by cytochalasin B treatment did not prevent subsequent PMA-stimulated neutrophil adherence. These results demonstrate that increased neutrophil adherence to surfaces can be induced by a nonchemotactic stimulus and that neutrophil adherence is independent of organized microfilaments.

Cell Adhesion↗
PubMed1987

Human monocyte adherence: a primary effect of chemotactic factors on the monocyte to stimulate adherence to human endothelium.

Monocyte emigration into areas of inflammation is initiated by monocyte adherence to the microvascular endothelium which may be induced by the local production of chemotactic factors at the inflammatory site. However, it is not clear whether such stimuli act on the monocyte and/or the endothelial cell to promote this effect. Accordingly, the effect of the chemotactic peptides C5a des arg and formyl-methionyl-leucyl-phenylalanine (FMLP) on human monocyte adherence to human microvascular endothelial cell monolayers was investigated in vitro. Monocytes (92 to 98% pure) were isolated by discontinuous plasma-Percoll density gradients and cell elutriation, methods designed to minimize monocyte exposure to endotoxin. Mean spontaneous (unstimulated) adherence of 111Indium-tropolonate-radiolabeled monocytes to microvascular endothelial cell monolayers was 19.7% +/- 1.3. Monocyte adherence to microvascular endothelial cell monolayers was stimulated in a dose-response fashion in the presence of C5a des arg or FMLP to a maximum mean adherence of 47.2% +/- 2.9 or 43.8% +/- 2.2, respectively. C5a des arg or FMLP stimulated monocytes to adhere to monolayers of human vascular smooth muscle cells, human dermal fibroblasts, or serum-coated plastic wells in a comparable fashion as to endothelial cells. The simultaneous presence of both chemotactic peptides C5a des arg and FMLP in the assay system stimulated monocyte adherence to the same degree as either stimulus alone. This finding suggested that those monocytes stimulated to adhere by C5a des arg were the same subpopulation responding to FMLP. Spontaneous monocyte adherence (in the absence of chemotactic peptides) to both endothelial cell monolayers and serum-coated plastic wells was reduced in the presence of plasma, but chemotactic peptides induced a significant, albeit reduced, adhesion of monocytes in this circumstance. The pretreatment of monocytes with either C5a des arg or FMLP prior to the adherence assay induced stimulus-specific desensitization of monocyte adherence. Neither a desensitization nor stimulated monocyte adherence occurred when endothelial cell monolayers or serum-coated plastic wells were pretreated with either of the chemotactic peptides. The fixation of endothelial cell monolayers prior to the adherence assay did not alter the degree of spontaneous, C5a des arg-stimulated, or FMLP-stimulated monocyte adherence. These data suggest that the stimulated adhesion of monocytes to endothelial cells by C5a des arg or FMLP represents primarily an effect of these chemotactic peptides on the monocyte.

Cell Adhesion↗