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Responses of the IgE type to various nuclear antigens have been explored using the human basophil degranulation test (HBDT) to single (SS) and double-stranded (DS) deoxyribonucleic acid (DNA), nuclear ribonucleoprotein (nRNP), and Sm antigens. This study was conducted on systemic lupus erythematosus (SLE) patients as compared to rheumatoid arthritis (RA) polymyositis (PM), scleroderma (SCL) and mixed connective tissue disease (MCTD). SLE and MCTD patients showed a high level of basophil degranulation to the four antigens. Degranulation to nRNP protein was higher in MCTD than in SLE cases. HBDT to the four antigens were constantly negative in control donors and in RA, PM and SCL. Basophil degranulation to DS-DNA in SLE was blocked by previous exposure of the blood to anti-human-IgE antiserum. These results exemplify the specificity of the HBDT, making this simple and fast test a useful tool in the diagnosis of connective tissue diseases.

The release of platelet-activating factor (PAF) from stimulated human endothelial cells (HEC) cultured from normal term, umbilical cord veins is described. HEC in primary cultures released PAF after challenge with A23187, rabbit anti-human factor VIII (RaHu/FVIII), angiotensin II, and vasopressin. HEC subcultures maintained the ability to release PAF in the presence of A23187 and RaHu/FVIII, whereas the release of PAF in response to angiotensin II and vasopressin was not constant and was reduced. Control cultured, smooth muscle cells derived from umbilical cord veins, previously depleted of endothelial cells, did not release PAF under the above-mentioned stimulation. Plastic-adherent or cultured monocytes released PAF with A23187, but not with RaHu/FVIII, angiotensin II, and vasopressin. The release of PAF from HEC in primary cultures required the presence of extracellular cations and the activation of membrane phospholipase A2. PAF release induced by A23187, RaHu/FVIII, angiotensin II, and vasopressin was unaffected by indomethacin, an inhibitor of cyclooxygenase, which, however, favored the release of PAF from HEC stimulated with thrombin, a stimulus that did not affect HEC in the absence of indomethacin. PGI2 inhibited PAF release from stimulated HEC. The relevance of an acetylation process in the biosynthesis of PAF and HEC was supported by the following evidence: 1) the increase in PAF yield in the presence of sodium acetate and, particularly, of acetyl-CoA; 2) the incorporation of [14C]acetate into PAF molecules; 3) the loss of radioactivity and of biologic activity after treatment with phospholipase A2. These results indicate that HEC in culture are able to release PAF and that metabolic pathways similar to those described for leukocytes are involved.

Triggering of large granular lymphocyte (LGL) Fc receptor with a specific monoclonal antibody (AB8.28) linked to an insoluble matrix induces cell activation, as witnessed by expression of HLA class II (DR and DQ) molecules and interleukin 2 receptor. Moreover, this event is accompanied by a concomitant release of platelet-activating factor by LGL. We conclude that the Fc receptor molecule identified by mAb AB8.28 represents a trigger for LGL activation.

Polymethylmethacrylate (PMMA) membranes with different net electric charges and percentage water contents (anionic 71%, neutral 70%, cationic 75%) were evaluated for their ability to stimulate plasma-free human polymorphonuclear neutrophils (PMN), and compared for potency to cuprophan (Cu), already described as being a potent trigger of PMN. The release of lysozyme, beta-glucuronidase, lactic dehydrogenase (LDH), and the generation of a platelet aggregating activity were studied in the supernatants from plasma-free human PMN incubated with different membranes. The PMN intracellular content of neutrophil cationic proteins (NCP), elastase, and cathepsin G were also studied by immunofluorescence using specific antisera on smears of PMN before and after incubation with each membrane. Only cationic, but not anionic or neutral PMMA induced a marked release of lysozyme (range 20-25% of the sonicated control, assumed as 100%), and beta-glucuronidase (40-43%), and marked depletion of the intracellular content of NCP, elastase, and cathepsin G, suggesting a degranulation process. Platelet aggregating activity was generated and referred to the release of platelet activating factor (PAF) only in the supernatants from PMN incubated with cationic, but not with anionic, or neutral PMMA membranes. These results indicate that modification of the net electric charge can per se turn PMMA, commonly recognized as inert, into a material with marked PMN activating effects, comparable to those of Cu, a highly reactive polymer.

Plasma-free, purified, normal, human polymorphonuclear neutrophils (PMN) were recirculated for 60 minutes in an experimental model of dialysis using cuprophan membranes and acetate or bicarbonate dialysate. At different time intervals, the intracellular contents of PMN-derived cationic proteins (NCP), the release of lysosyme, beta-glucuronidase and PAF as well as the occurrence of PMN and platelet aggregating activities in the supernatants were evaluated. The formation of PMN aggregates, the depletion of intracellular contents of NCP together with the release of lysosomal constituents occurred early (5-10 min) in the course of recirculation. These events were concomitant with the occurrence of PMN aggregating activity in the supernatants due to the release of NCP, as it was antagonised (30-40%) by a rabbit anti-human NCP, and to the release of PAF which also accounted for the platelet aggregating activity that was independent from both adenosine diphosphate and cyclo-oxygenase inhibitors. These data suggest that direct interaction occurs between human PMN and cuprophan in in vitro conditions in the absence of plasma factors and point to a role for cellular mediators in the pathogenesis of the intravascular alterations occurring early in haemodialysis.

During "hyperacute rejection" of rabbit heart perfused with transplantation alloantibodies, platelet activating factor (PAF) was released into the coronary effluent, which appeared to have physicochemical and functional properties similar to the 1-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (synthetic PAF) and to PAF obtained from IgE-sensitized rabbit basophils. The release of PAF was associated with an early tachycardia, followed by increasing bradycardia and conduction arrhythmias, as well as decrease of coronary flow and of amplitude of electrogram. The heart stopped beating within 30 min. The release of PAF as well as the "rejection" required the presence of fresh rabbit serum as a source of complement. The PAF receptor antagonist SRI 63-072 in a dose of 0.6 mg, reversed by 70% the reduction of coronary flow within 2-4 min after its addition to the perfusate; ED50 was 0.4 mg. Bradycardia and arrhythmia were reduced; however, the normal electrical activity was only occasionally restored. The cessation of heart action was delayed up to 50 min after the beginning of perfusion with transplantation alloantibodies and complement, but it was not prevented. These results suggest that PAF is released during "rejection" of the heart perfused in vitro with serum containing transplantation alloantibodies in the absence of inflammatory cells and that this mediator is at least in part responsible for the deterioration of cardiac function.

Platelet-activating factor (PAF) is released in vitro during human and rabbit polymorphonuclear neutrophil (PMN) aggregation induced by C5a anaphylatoxin, neutrophil cationic proteins (CP) and their carboxypeptidase-B-derived fragments, C5a des Arg and CP des Arg, as well as phagocytosis of opsonized baker's yeast particles and immune complexes (IC). Purified PAF itself is able to cause in vitro PMN aggregation. By using selective inhibitors, we show that PMN aggregation, induced either by PAF or by other soluble stimuli such as C5a, CP and their des Arg products, follows a similar metabolic pathway, which is both adenosine-diphosphate-(ADP)- and arachidonic acid (AA)- independent. The in vivo injection of purified PAF into rabbits leads both to formation of intravascular PMN aggregates and to development of acute neutropenia, which has the same features as those observed after challenge with IC, C5a and CP. In this respect, electron-microscopic studies of intravascular PMN aggregates in the pulmonary capillary network and glomeruli show identical ultrastructural patterns. Moreover, the intravascular release of PAF is demonstrated after the intravenous injection of IC and temporally correlated with the development of neutropenia. We suggest that PAF is probably the final, common, effector substance of IC-, C5a-, C5a-des-Arg-, CP-, CP-des-Arg-mediated PMN aggregation.

The localization of cationic proteins (CP) derived from platelets and from polymorphonuclear neutrophils (PMN) in glomeruli of 42 rabbits injected i.v. with a large amount of bovine serum albumin, was investigated in sequential biopsies by immunofluorescence, using goat-anti-platelet CP and anti-PMN CP sera. Platelet CP deposits became detectable within 7 to 8 days after the i.v. injection of bovine serum albumin, before or coincident with the onset of proteinuria. The intensity and the extent of linear and segmental deposits of platelet CP along the glomerular capillary walls reached a peak at day 9 to 10, when proteinuria was maximal. The anti PMN-CP serum stained the cytoplasm of the few PMN present in glomeruli and only occasionally at day 11 and 12 identified focal deposits of PMN-CP along the glomerular capillary walls. The kinetic study of glomerular immune deposits showed that the first appearance of immune deposits in antigen excess was preceded by, or was concomitant, with the detection of platelet-CP in glomeruli. In the later stages of serum sickness, the immune deposits showed a progressive increase in rabbit IgG and C3. The glomerular polyanions were studied by light microscopy, using the colloidal iron technique, and by electron microscopy using polyethyleneimine as a cationic probe. The glomerular deposits of platelet-CP were associated with a reduction of colloidal iron staining, which was maximal 9 to 11 days after the i.v. injection of bovine serum albumin. At day 15, colloidal iron staining was almost completely restored. At day 9 in rabbits with acute serum sickness the anionic sites of glomerular basement membrane evidenced by polyethyleneimine, were segmentally decreased, mainly in the lamina rara interna. In rabbit studied at day 15 the anionic sites were decreased only at the base of the subepithelial electron dense deposits (humps). These results suggest that in rabbits with experimentally-induced acute serum sickness, during the early stages of glomerular immune deposit formation endogenous CP, released mainly from platelets, bind to glomerular capillary walls and possibly contribute to the neutralization of glomerular polyanions.

The urinary protein excretion rate, the glomerular localization of cationic proteins (CP) derived from platelets and polymorphonuclear neutrophils (PMN) and the loss of fixed anionic charges were studied in rabbits infused with synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine [platelet activating factor (PAF), 1.5 micrograms/kg in 2 ml of saline containing 0.25% bovine serum albumin (BSA)]. The urinary protein excretion rate, unaffected by diphenhydramine, an antihistaminic agent, reached its maximum at 180 min and decreased 24 hr after PAF infusion. The localization of CP derived from platelets and PMN was investigated by immunofluorescence using specific antisera. Platelet-derived CP were detectable in glomeruli at 15 min and, particularly, at 180 min after PAF infusion. Cells positive for CP derived from PMN accumulated within 15 min in the glomerular capillaries and, later (180 min), cytoplasmic depletion and localization in glomerular capillary walls occurred. CP deposits were associated with the loss of fixed anionic charges as detected by ruthenium red and colloidal iron staining. Rabbits infused with 1-0-octadecyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF) or saline-BSA alone had none of the alterations described above. The development of proteinuria, the glomerular localization of platelet- and PMN-derived CP and the concomitant loss of fixed anionic charges suggested the possibility that, once CP were released in the circulation from PAF-stimulated platelets and PMN, they bound to, and neutralized, fixed anionic charges, resulting in enhanced glomerular permeability.

The intratracheal instillation into rabbits of 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) or native platelet-activating factor (PAF) was shown to induce a dose-dependent acute pulmonary inflammation characterized by accumulation of macrophages in the alveolar space, degenerative and necrotic changes of alveolar epithelium, and accumulation of polymorphonuclear leukocytes (PMNs) and platelets in the alveolar capillary lumens with degenerative changes of endothelial cells. Infiltration of alveolar septa by inflammatory cells and, in a later stage, pulmonary fibrosis were also observed. Intrabronchial instillation of lysoglyceryl ether phosphorylcholine (lyso-GEPC) produced no inflammatory changes or only mild ones. In comparison with acute inflammation induced by intratracheal instillation of C5a des Arg, which is mainly characterized by the presence of neutrophils, red blood cells, and fibrin in the alveolar space, AGEPC and native PAF seem to induce a more severe accumulation of macrophages in the alveolar space and septa and of platelet and PMNs in the lumens of alveolar capillaries. These results are compatible with the concept that during inflammatory reaction an intraalveolar release of PAF contributes to the development of pulmonary injury.

Synthetic polycations have been shown to bind and neutralize glomerular polyanions (GPA), thereby increasing the permeability of the glomerular capillary wall (GCW). In the present study it is demonstrated that human platelet-derived cationic proteins (HuPlt CP), which are able to increase cutaneous vascular permeability, bind in vitro to the GCW following incubation of normal human kidney sections with purified HuPlt CP or with washed human platelets stimulated with thrombin, immune complexes (IC) and platelet-activating factor (PAF), or stimulated with a suspension of washed human platelets and polymorphonuclear leukocytes in the presence of phagocytable substrate. The antiserum used in immunofluorescence test to detect the binding of HuPlt CP was specific for two different molecular types of HuPlt CP, both with an isoelectric point (pI) of 10.5. Glomerular deposits of HuPlt CP were also detectable by immunofluorescence microscopy in renal glomeruli present in tissue obtained by biopsy from patients with systemic lupus erythematosus (SLE), a disease in which platelets have been implicated as mediator of glomerular injury. These data indicate that when activated platelets release HuPlt CP in vivo, these proteins bind to glomerular structures. The binding of HuPlt CP to GCW appears to be ionic in nature since heparin, a polyanion, prevents this binding in vitro. In addition, heparin, as well as a high molarity buffer, removed deposits of HuPlt CP bound in vitro to normal GCW or bound in vivo to glomeruli of patients with SLE. The binding of HuPlt CP to GCW is associated with loss of colloidal iron staining, a qualitative technique that demonstrates primarily epithelial cell surface anionic sialoglycoproteins. In experiments of in vitro binding of purified HuPlt CP to section of normal kidney treatment with heparin completely restores the normal pattern of colloidal iron staining suggesting ionic neutralization of GPA. In contrast, heparin is only partially effective in restoring colloidal iron staining in normal kidney sections treated with platelets directly stimulated with IC or PAF or in kidney sections of patients with SLE. These observations indicate that under these conditions the ionic interaction of HuPlt CP with GCW is only partially responsible for the loss of colloidal iron staining. The results of the present study suggest that biologically active polycationic mediators released from stimulated platelets localize in GCW and participate in the induction of glomerular injury.

Soluble and phagocytic stimuli released PAF-acether from PMN leucocytes, as determined by chromatography and bioassay by platelet aggregation. The same material caused aggregation of human and rabbit PMN leucocytes in vitro which was inhibited by ETYA and PGI2. PGI2 also inhibited PAF-acether release by PMN leucocytes and, in vivo, PGI2 abolished not only PAF-acether-induced, but also immune complex or C5a-induced thrombocytopenia and neutropenia in rabbits. These data suggest that PAF-acether may be involved in activation of both platelets and PMN leucocytes in vivo.

We determined in normal nonpregnant (group 1) women, normal pregnant (group 2) women, and patients with preeclampsia (group 3) the serum inhibition of platelet-activating factor activity, the presence of detectable amounts of platelet-activating factor in the blood, and platelet responsiveness in vitro to platelet-activating factor, and to other agonists (adenosine diphosphate, collagen, and ristocetin), and prostacyclin (prostaglandin I2). In patients with preeclampsia (group 3) the serum inhibition of platelet-activating factor activity was significantly lower than that in groups 1 and 2. However, no detectable amounts of platelet-activating factor were observed. The mean values of platelet aggregation induced by platelet-activating factor, adenosine diphosphate, collagen and ristocetin, and the prostaglandin I2-inhibitory concentration of 50% which is inversely correlated with platelet sensitivity to prostaglandin I2, were not significantly different between groups 2 and 3. It is suggested that in preeclampsia the defect in serum inhibitory potential of platelet-activating factor--induced platelet aggregation may contribute to the disturbance in the homeostatic balance between proaggregant and antiaggregant substances.

Deposits of C3 but not of C1q and C4 were detected on the proximal tubules of kidneys from nephrotic patients with non-selective proteinuria. The incidence of tubular C3 deposits was significantly higher in patients with membranous glomerulonephritis, focal glomerulosclerosis, membrano-proliferative glomerulonephritis and non-selective proteinuria than in patients with minimal change disease, nephrotic syndrome and selective proteinuria or in patients with glomerular disease, but without nephrotic syndrome. The occurrence of tubular C3 deposits was positively correlated with the amount of urinary C3 excretion. In vitro studies showed that the human normal kidney as well as pathologic specimens negative for in vivo tubular C3 deposits were able to bind C3 on the brush border of proximal tubules when incubated with fresh heterologous serum. In contrast, in patients with non-selective proteinuria and in vivo tubular C3 deposits, the binding of heterologous C3 to the brush border of proximal tubules was markedly reduced. The positive correlation between the occurrence of tubular C3 deposits and the urinary complement excretion, together with the detection of the C3 breakdown products in the urines further supported the hypothesis that complement components, once filtrated through the glomerular barrier, might be activated by the brush border of the proximal tubule.

Monocyte-derived interleukin 1 (IL 1) induced the platelet activating factor (PAF) production in cultured human endothelial cells (HEC). The product was identified as PAF by the chemical properties, the susceptibility to phospholipase A2 and C and to lipase A1, its behaviour in thin layer chromatography and in high pressure liquid chromatography and the recovery of biological activity tested on rabbit platelets. The action of IL 1 was concentration-dependent and took more than 2 hours to became apparent. Most of the PAF produced was cell-associated and only the 25% of the total was released.

In spite of the anticoagulant activity of heparin platelet deposition and contact activation of coagulation occurs during dialysis. We have studied platelet counts, fibrinogen, platelet factor 4, beta-thromboglobulin, thromboxane B2, FRA, C3d and kallikrein values, whole blood and euglobulin lysis times and membrane areas in haemodialysis using cuprophan and cellulose acetate and in haemofiltration with polyacrilonitrile. Deposits on all the three membranes included leucocytes, platelets and fibrin. The coagulation and fibrinolytic systems are activated more intensively with cellulose acetate and more prolongedly with polyacrilonitrile. Platelet factor 4 and beta-thromboglobulin increases suggest platelet activation, only partially dependent on arachidonic acid-mediated pathway as thromboxane B2 is not increased. The complement system is activated whereas serum kallikrein does not alter, suggesting that platelets rather than factor XII are crucial in contact activation.

TNF and IL-1 stimulate the synthesis and release of platelet-activating factor (PAF) by neutrophils and vascular endothelial cells. Serum inhibits PAF production even after inactivation of an acetylhydrolase that degrades PAF. Human plasma was fractionated by gel filtration chromatography, and two inhibitory fractions were detected, one containing PAF-acetylhydrolase activity and the other alpha 1-proteinase inhibitor. Low concentrations of this antiproteinase and of human plasma alpha 1-antichymotrypsin inhibited TNF-induced PAF synthesis in neutrophils, macrophages, and vascular endothelial cells. Both antiproteinases also inhibited PAF production stimulated by phagocytosis in macrophages and induced with IL-1 in neutrophils or with TNF in vascular endothelial cells. These results suggest that a proteinase activated on the plasma membrane or secreted by these cells is involved in promoting PAF synthesis. Indeed, addition of elastase to macrophages, neutrophils, and endothelial cells stimulated synthesis and release of PAF much faster than TNF. A similar stimulation was observed in incubations with cathepsin G. To identify a proteinase activated in TNF-treated cells, neutrophils and endothelial cells were incubated with specific chloromethyl ketone inhibitors of elastase and cathepsin G. Synthesis of PAF was significantly inhibited by low concentrations of the cathepsin G inhibitor. The finding that antiproteinases are inhibitory at concentrations 100-fold lower than those present in plasma raises questions as to the ability of TNF and IL-1 to stimulate neutrophils in circulation or endothelial cells to synthesize PAF. We propose that PAF production is limited to zones of close contact between cells, which exclude antiproteinases.

Several findings reveal the involvement of the IgE-basophil-mastocyte-platelet-activating-factor (PAF) system in human acute post-streptococcal glomerulonephritis. In the acute phases of the disease there is a transient, marked reduction in the circulating metachromatically staining basophils, indicating an in vivo basophil degranulation. The blood reservoirs of PAF are depleted. The number of metachromatic mastocytes in renal biopsy samples was very low and morphological aspects of degranulation were present. In vitro, we demonstrated basophil degranulation and PAF release in presence of exogenous streptococcal Ags after recovery. These findings suggest that the IgE-basophil-mastocyte-PAF system may play a role in human pathology, as has been shown in immune complex (Ic) deposition in acute serum sickness in rabbits.

A simple immunohistological test has been developed to detect and to quantitate the presence of immune complexes (IC) on the surface of human polymorphonuclear neutrophils (PMN). The interaction between IC and PMN has been evaluated in several human IC diseases. High amounts of immunoglobulins (Ig) and C3 were detected on the PMN surface from these patients. The deposits of Ig and C3 were inversely related to the percentage of membrane-free receptors for Fc and C3.

The macroscopic signs of rejection, the levels of circulating transplantation antibodies, the histologic and immunocytochemical aspects, and the levels of platelet-activating factor (PAF) present in the venous blood were studied in two groups of rabbits that had received renal allografts, one group presensitized with multiple skin grafts and a second group unsensitized, as well as in a third group of rabbits that had received renal autografts. All of the eight allografts hyperacutely rejected by presensitized recipients had deposits of rabbit IgG, IgM, and C3 along the endothelia of the vessels and massive intravascular accumulation of platelets (Pt) as soon as 5 minutes after revascularization. PAF release was detected in 2 to 10 minutes after revascularization and was present throughout most of the 60 minutes of observation. Sixty minutes after transplantation Pt and polymorphonuclear leukocytes (PMN) obliterated the vasculature and deposits of Pt- and PMN-derived cationic proteins were detected in the lumina and in the walls of the capillaries. Similar, but less severe, findings were observed in three of six renal allografts which had transient episodes of rejection after transplantation into presensitized recipients. In contrast, circulating transplantation antibodies, macroscopic signs of rejection, vascular immune deposits, release of PAF, and microvascular thrombosis were not detected in renal allografts transplanted into unsensitized recipients or in renal autografts. The results indicate that in hyperacute renal allograft rejection there is an immediate fixation of transplantation antibodies and complement of the recipient to endothelial antigens of the graft, local release of PAF, and massive accumulation, aggregation, and degranulation of Pt and PMN in the vasculature, resulting in a binding of Pt- and PMN-derived cationic proteins to the walls of the capillaries. It is conceivable that PAF release and Pt and PMN cationic proteins may contribute, together with other lysosomal enzymes, vasoconstriction, and coagulation, to the pathogenesis of antibody- and complement-mediated hyperacute graft injury.

This study shows that platelet-activating factor (PAF) is released in significant amounts (10.9 +/- 12.8 ng/min) during the initial reperfusion of the ischemic-isolated rabbit heart. When reperfusion was performed in the presence of autologous rabbit platelets, the electrical and mechanical alterations characteristic of this phase were significantly worsened. These alterations were antagonized by pretreatment of rabbit platelets with a PAF receptor antagonist (SDZ 63-675), suggesting a contribution of PAF released during reperfusion in the cardiac dysfunction. To discriminate whether the effect of PAF was platelet dependent, infusion of PAF (10-40 ng) was performed in nonischemic rabbit hearts perfused with or without autologous platelets. Although the effect of PAF per se was minimal, in the presence of platelets PAF induced a biphasic effect characterized by a transient positive inotropism followed by a dose-dependent decrease in coronary flow, negative inotropism, reduction of action potential duration, and conduction arrhythmias. These effects were abolished by pretreatment of platelets with SDZ 63-675, suggesting a PAF receptor-mediated platelet activation. In addition, a relative contribution of histamine, thromboxanes, and leukotrienes released from activated platelets was inferred by experiments performed with pyrilamine and cimetidine, imidazole, and FPL 55712, respectively.

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1-100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC50 was 1 nM. The response to oxytocin (0.01-10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desensitization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.

Basophil degranulation and release of platelet-activating factor (PAF acether) have been implicated in enhanced vascular permeability and immune complex deposition in rabbit acute serum sickness. PAF-acether is a phospholipid mediator of anaphylaxis, released from leukocytes of several mammalian species, including man, that aggregates platelets and releases their vasoactive amines. In this article, we evaluated the occurrence of basophil degranulation and release of PAF-acether in patients with systemic lupus erythematosus. In the acute phases of the disease, basophils were in vivo degranulated, and the amount of PAF-acether releasable from leukocytes was markedly reduced. During remission or in the latent phases of the disease, when the number of metachromatically staining basophils increased. in an vitro degranulation and release of PAF-acether were observed after DNA challenge. Electron microscopy studies demonstrated that basophils indeed degranulated in response to DNA. These studies also showed the interaction between degranulating basophils and human platelets which aggregated even if the other two pathways of human platelet aggregation, i.e., the ADP- and the arachidonic acid-dependent pathways, were blocked. The concomitance of basophil degranulation and release of PAF-acether, together with the morphologic evidence of the interaction between degranulating basophils and aggregated platelets, was strongly suggestive of release of PAF-acether from basophils in systemic lupus erythematosus.

In the present study it is demonstrated that platelet-activating factor (PAF) was produced by chick retinas, upon stimulation with neurotransmitters such as acetylcholine (ACh), dopamine, or with calcium ionophore A23187, but not upon stimulation with gamma-amino-n-butyric acid, L-glycine, L-glutamate, epinephrine, or histamine. PAF produced in response to ACh, dopamine, or A23187 was not released into supernatants but was extractable from retinas. The amounts of extractable PAF increased after sonication of stimulated retinas. While no PAF activity could be recovered from unstimulated retinas, small amounts of this lipid can be detected following sonication of the tissue. The amount of extractable PAF from ACh-, dopamine-, or A23187-stimulated retinas was dependent upon the incubation time and concentration of the agonists. PAF was identified on the basis of chemical and lipase treatments, biological activity with washed rabbit platelets, behavior on thin layer chromatography, and high pressure liquid chromatography. Control cell preparations (leukocytes, erythrocytes, and embryogenic fibroblasts) did not produce PAF upon neurotransmitter stimulation. ACh and dopamine promoted PAF production by increasing dithiothreitol-insensitive cholinephosphotransferase activity, without affecting the acetyltransferase activity. In contrast, the A23187 ionophore stimulated the acetyltransferase activity but did not affect the dithiothreitol-insensitive cholinephosphotransferase.

The cationic protein (CP) content of polymorphonuclear neutrophils (PMN) prepared for transfusion is depleted after storage. The supernatants from these PMN have in vitro a PMN aggregating activity which is abolished by the preabsorption with a specific rabbit anti-human PMN CP serum. Furthermore, when the supernatants stored for few hours were injected into New Zealand White rabbits, a marked sequestration of PMN took place in the lung microvascular bed. It is suggested that PMN storage per se can cause the release of intracellular mediators of possible pathogenetic importance in the development of the pulmonary infiltrates observed after PMN transfusions.

The binding of human platelet cationic proteins (HuPlt CP) to rat renal cortex in vitro and in vivo, the loss of glomerular polyanions (GPA) and the increase in glomerular permeability were studied. HuPlt CP were purified by sequential cation-exchange chromatography and chromatofocusing, by which these proteins were shown to be highly cationic in nature (pI 10.5) and mainly composed of three molecular species of 55.60 kD, 40.45 kD, and 10 kD as studied by gel permeation in high pressure liquid chromatography and SDS-polyacrylamide gel electrophoresis. Binding of HuPlt CP to glomerular capillary walls (GCW), mesangium and to peritubular capillaries of the rat renal cortex was demonstrated by immunofluorescence, using a specific goat anti-HuPlt CP antiserum, after incubation of the sections with HuPlt CP in vitro and after injection of HuPlt CP in vivo. This interaction was ionic in nature, since treatment of sections with heparin abrogated the binding of HuPlt CP to glomerular structures. The glomerular deposits of HuPlt CP were associated with the loss of GPA as revealed by colloidal iron staining (light microscopy) in both in vitro and in vivo experiments and by ruthenium red staining (electron microscopy) in in vivo studies. After the injection of native ferritin, the increase in glomerular permeability produced by an infusion of HuPlt CP was observed by the increased ratio of counted particles within the glomerular basement membrane with respect to controls. The binding of HuPlt CP to GCW and the loss of GPA was consistent with the interpretation that HuPlt CP may increase glomerular permeability due to the neutralization of GPA.

Synthetic platelet-activating factor (PAF) (1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) has been shown to induce a slowly developing contraction of rabbit lung parenchymal strips in an isolated organ bath. The spasmogenic effect of AGEPC appeared to be mediated by specific receptors distinct from H1, H2, cholinergic and C5a anaphylatoxin receptors. Prior exposure to AGEPC induced specific desensitization of lung parenchymal strips. Experiments with several pharmacological agents indicated that AGEPC-induced contraction was independent from cyclooxygenase, but was blocked when phospholipase A2 and lipoxygenase were inhibited and when the Ca++ channels were antagonized. Corticosteroids exhibited an inhibitory effect specific for AGEPC. Intracellular levels of cyclic AMP or cyclic GMP seemed to have a modulatory role in AGEPC-induced contraction of rabbit lung parenchymal strips.

This study reports the results of in vitro investigations on the aggregation of polymorphonuclear neutrophils (PMN) induced by the C5a anaphylatoxin complement component as well as the cationic proteins (CP), which are released by challenging PMN with immune complexes (IC). The carboxy-peptidase-derived des-Arg fragments of CP and C5a; CPi and C5ai, inactive in terms of anaphylactic and chemotactic activity, nevertheless showed a more potent ability to aggregate PMN than CP and C5a. The process of PMN aggregation required metabolic energy and divalent cations, Ca++ and Mg++. The microtubular system and the subplasmalemmal microfilaments appeared to be of critical importance. Electron microscopic studies on aggregates of PMN obtained on stimulation with CP, C5a, CPi and C5ai showed parallel tracts of variable length of cell membranes at the points where cells were in contact with each other.

Monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of platelet-activating factor (PAF) in cultured human vascular endothelial cells (HEC). The product was identified as PAF by its behavior in chromatographic systems, its recovery of biological activity, and its physico-chemical properties and susceptibility to lipases. The response of HEC to IL-1 was concentration-dependent, took more than 2 h to become apparent, and decreased after 18 h of incubation. Most of the PAF produced was cell-associated and only a small amount (about 25% of the total) was released in the culture medium. To study the mechanism of IL-1-induced HEC-PAF production we tested the activity of 1-O-alkyl-sn-glycero-3-phosphocholine:acetyl/coenzyme A acetyltransferase in HEC. Acetyltransferase activity measured in IL-1-stimulated HEC lysates showed a three to five times greater maximum velocity, but the same Michaelis constant, as untreated cells. The regulation of PAF generation in HEC by IL-1 may be an important aspect of the two-way interaction between immunocompetent cells and vascular tissue.

Several immunological mechanisms of platelet involvement in inflammation are described. Basophils degranulate and release a platelet-activating factor (PAF) when challenged with anaphylotoxins, cationic proteins (CP) from polymorphonuclear cells (PMN) as well as with specific antigens from atopic patients. PAF is a newly-discovered mediator of anaphylaxis present in rabbit and human basophils. This factor is a small phospholipid, probably a lyso-1-phosphatidylcholine with a significant aggregating activity on rabbit and human platelets. PAF is released, together with a small amount of arachidonic acid, from basophils in the presence of anaphylotoxins, CP and specific antigens from atopic patients. The already well-known direct interaction between immune complexes (Ics) and human platelets is compared here with the mechanism of PAF-induced aggregation. It is shown that the latter process of aggregation differs from the former. Human platelet aggregation starts more rapidly in the presence of PAF than of Ics. PAF-induced aggregation is ADP-independent as it is not affected by ADP inhibitors. On the contrary, Ic-dependent aggregation is brought about by endogenous ADP release and therefore inhibited by ADP inhibitors. The interaction between Ics and platelets leads to the release of CP. The latter produce a cascade reaction involving basophils which degranulate and release PAF. A self-maintaining mechanism of tissue injury is thus triggered.

CB04 monoclonal antibody which reacts with an epitope of a surface molecule expressed on human monocytes has been elicited using peripheral blood lymphocytes as immunizer. The characterization of the monoclonal antibody examined at the phenotypic, molecular, and functional levels indicates that the CB04 antibody defines a structure present on monocytes, tissue macrophages, B cells, polymorphonucleates, and erythrocytes. The molecular weight (220 kD), the tissue distribution in health and disease conditions, and the involvement in relevant biological processes indicate that the CB04 structure is the receptor for the C3b fragment of the complement. The binding of the antibody to the cell surface induces inhibition of the C3bi receptorial function.