Effect of dialyzer reuse on complement activation and neutropenia in hemodialysis.

A prospective study of 10 patients undergoing hemodialysis showed that less neutropenia and complement activation occurred with dialyzer reuse. Neutrophil counts fell 95% +/- 5% (SEM) with first use and 66% +/- 8% and 48% +/- 10% with second and third uses, respectively (p less than 0.05). The production of complement component C5a-desarg, as measured by the bioassay granulocyte aggregation, was decreased by 96% +/- 1% and 93% +/- 2% with second and third uses, respectively (p less than 0.05). We investigated the role of the dialyzer disinfectant formaldehyde in decreased neutropenia. In vitro, formaldehyde inhibited granulocyte aggregation and chemotaxis and the dialyzer membrane's ability to generate granulocyte aggregating activity; however, this occurred only at concentrations higher than those likely to obtain in patients. The ability of dialysis membranes to generate granulocyte aggregating activity in plasma was decreased 55% +/- 5% by their prior sequential preincubation in plasma and then formalin (p less than 0.05) and extensive rinsing, which is similar to the circumstances obtaining with dialyzer reuse. Preincubation of membranes in plasma or formalin alone resulted in no change in the membrane's ability to generate granulocyte aggregating activity. We conclude that the exposure of membranes to both plasma and formalin during dialysis and storage is responsible for the decreased C5a-desarg production with reuse, probably because plasma proteins are fixed to the membrane in such a way that interrupts free interaction between the membrane and plasma complement components.

Ibuprofen inhibits granulocyte responses to inflammatory mediators. A proposed mechanism for reduction of experimental myocardial infarct size.

The use of nonsteroidal antiinflammatory agents to reduce myocardial infarct size has demonstrated a dichotomy between ibuprofen, which reduces myocardial infarct size, and aspirin, which does not. A feline model of coronary ischemia using ligation of the anterior descending artery demonstrated that intravenous ibuprofen (2.5-20 mg/kg) given immediately and 2 h after ligation significantly decreased (by about 40%) myocardial infarct size. In contrast, aspirin did not diminish infarct size at any achieved dose; in fact, at some doses it tended to increase infarct size. In vitro studies with purified granulocytes demonstrated a similar dichotomy between ibuprofen and aspirin. Ibuprofen inhibits granulocyte aggregation, superoxide production, lysosomal enzyme release, and granulocyte-mediated endothelial cytotoxicity, while aspirin is without effect on these modalities. We propose that ibuprofen's beneficial effect in experimental myocardial ischemia is related to its ability to inhibit activated granulocytes and thus to diminish myocardial cell death in experimental myocardial infarction.

Complement, granulocytes, and shock lung.

Intravascular leukostasis in the pulmonary microvasculature is a cardinal early histologic finding in patients with shock lung. Identical leukostasis is also observed in patients undergoing extracorporeal hemodialysis with cellophane membrane dialyzers, and it has been documented that the accumulation of granulocyte plugs in the lung is mediated by complement activation triggered by dialyzer cellophane. The C5a-desarg so generated causes peripheral blood granulocytes to aggregate, and the aggregates so formed embolize to the lung, where they cause occlusion of the microvasculature and increased capillary leakage, manifested by interstitial and alveolar edema. In vitro studies suggest that this endothelial damage is mediated by hydrogen peroxide from the adherent granulocytes. Most importantly, a close correlation has been found between the presence of C5a-desarg in plasma and the subsequent onset of shock lung in patients after trauma, burns, and sepsis. As exemplified by hemodialysis leukopenia, C5a-desarg-mediated pulmonary leukostasis is a self-limiting process because of selective down-regulation of granulocyte receptors for C5a-desarg--a mechanism that primarily limits the lung damage associated with intravascular complement activation.

Chronic myelogenous leukemia. Development of blast crisis with both lymphoid and myeloid features.

A 50-year-old man had chronic myelogenous leukemia and entered a blast crisis that was both morphologically and histochemically lymphoid. The blasts contained terminal deoxyribonucleotidyl transferase and expressed lymphoblastic leukemia-associated antigen. He rapidly entered remission with vincristine sulfate and prednisone therapy. Nevertheless, his blasts displayed a marker generally considered unique to myeloid cells: they selectively bound the granulocyte chemotaxin N-formyl-Met-Leu-Phe. In addition, some cells contained granules resembling those of basophils or mast cells. Such mixed myeloid-lymphoid features in chronic myelogenous leukemia blast cells may reflect malignant transformation of a stem cell capable of both myeloid and lymphoid differentiation, or they may reflect the dedifferentiation as a feature of malignant change.

Limbic encephalopathy as a nonmetastatic complication of oat cell lung cancer. Its reversal after treatment of the primary lung lesion.

A 52-year-old white woman presented with dementia, a seizure disorder, and an inappropriate affect characteristic of limbic encephalopathy. Chest x-ray showed a mass lesion that, on biopsy, proved to be oat cell carcinoma. Her central nervous system symptoms improved following radiotherapy limited to the primary lesion and later resolved completely with attainment of a complete remission after chemotherapy with cyclophosphamide, doxorubicin, vincristine, and VP-16--drugs unable to penetrate the cerebrospinal fluid. The resolution of the paraneoplastic syndrome in this patient without the addition of cranial irradiation suggests that a trial of cytoreductive therapy is warranted in patients with limbic encephalopathy associated with an underlying neoplasm.

Zymosan activation of plasma reduces hypoxic pulmonary vasoconstriction.

Small doses of endotoxin (15 micrograms/kg IV) inhibit the pulmonary vascular pressor response to alveolar hypoxia in the anesthetized dog. One of the actions of endotoxin is to initiate the alternate pathway of complement activation. Incubation of human plasma with zymosan (ZAP) will activate this pathway. We wished to see if ZAP would mimic the effect of endotoxin. Prior to ZAP, hypoxia (F1O2 12%) in 5 anesthetized dogs increased pulmonary vascular resistance (PVR: mm Hg/L/min) from 3.7 +/- 0.8 to 7.1 +/- 1.5. After 50 ml ZAP IV the PVR change with hypoxia was only from 3.6 +/- 0.6 to 3.9 +/- 0.8. Plasma heated to destroy complement prior to ZAP incubation in one experiment did not reduce the pressor response. In a further 5 dogs pretreated with meclofenamate (2 mg/kg IV) the PVR increased from 3.7 +/- 0.4 to 7.5 +/- 0.4 with hypoxia prior to IV ZAP and from 4.4 +/- 0.5 to 6.5 +/- 0.6 after ZAP. The effect of ZAP indicates that endotoxin may work through the activation of complement. The protection of the hypoxic pressor response by meclofenamate suggests that the ZAP inhibition (like endotoxin inhibition) may involve dilator prostaglandin-like substances.

Glycogenolysis versus glucose transport in human granulocytes: differential activation in phagocytosis and chemotaxis.

Granulocytes depend primarily on anaerobic glycolysis to supply the necessary energy for locomotion and chemotaxis. Either transmembrane transport of extracellular glucose or catabolism of intracellular glucose can supply glycolytic substrate. In this report, using enzymatic analysis of granulocyte glycogen, we describe conditional requirements for glycogenolysis, namely phagocytosis. With abundant extracellular glucose, granulocyte glycogen content (12.2 +/- 1.6 micrograms/10(6) cells) is not depleted whether or not incubations include various soluble chemotaxins (e.g., FMLP, C5ades arg, arachidonic acid). These chemotaxins accelerate transmembrane glucose uptake. With near complete absence (less than 6 mg/dl) of extracellular glucose, both resting and chemotaxin (FMLP, C5ades arg, arachidonic acid) stimulated granulocytes catabolize significant endogenous glycogen. Phagocytosis, however, fails to enhance glucose uptake and promotes glycogen consumption even with abundant extracellular glucose. Simple particle-phagocyte attachment without internalization (produced by cytochalasin-B) also promoted glycogen consumption, suggesting that this membrane deformation alone is a sufficient trigger for glycogenolysis. Resting or chemotactic granulocytes, therefore, can adapt their energy source pending extracellular glucose availability--often compromised at inflammatory sites-while phagocytic cells depend primarily, if not exclusively, on endogenous glycogen stores. This differential metabolic activation defends the granulocytes energy supply and may be critical in supporting antimicrobial activity in acute inflammation.

Granulocytes utilize different energy sources for movement and phagocytosis.

Granulocytes depend on anaerobic glycolysis for the energy required for chemotaxis, phagocytosis, and microbial killing. Two potential sources of the needed glucose are available: exogenous glucose and intracellular glycogen. These studies demonstrate that chemotaxin-induced movement of granulocytes induces accelerated uptake of exogenous glucose while phagocytosis does not, presumably utilizing instead the relatively slow process of glycogenolysis. As measured by incorporation of extracellular radiolabeled hexoses [1-14C]glucose or [3H]deoxyglucose), the soluble chemotaxin-aggregants of granulocytes, nF-met-leu-phe, C5ades arg, bacterial filtrate, or arachidonic acid all augment transmembrane hexose uptake. This insulin-like activity closely parallels the dose-related effects of these agents on induction of granulocyte aggregation and chemotaxis. Insulin, itself, affects glucose transport minimally and mainly at supraphysiologic concentrations. In contrast, phagocytic stimuli fail to enhance hexose uptake at all, despite stimulating catabolism of glucose, which in turn is probably generated by glucogenolysis. these data show that granulocytes, whose motile function occurs in glucose-rich milieu, alter in tandem their cellular glucose uptake with their movement response. For phagocytosis, which often occurs in hypoglycotic, purulent exudates, granulocytes depend on stored energy supplies--probably glycogen. This coordination may be crucial in supporting granulocyte antimicrobial activity during acute inflammation.

Activation of plasma complement by perfluorocarbon artificial blood: probable mechanism of adverse pulmonary reactions in treated patients and rationale for corticosteroids prophylaxis.

Perfluorocarbons have shown promise as clinical blood substitutes. Although early experience in Japan with one such product--Fluosol-DA--has been uncomplicated, we observed an adverse pulmonary reaction in the first American patient to receive it and know of similar reactions in two other Americans so treated. Postulating that activation of plasma complement (C) by the perfluorocarbon emulsion might have caused the reaction, we tested the product to determine if it is an activator of complement. Incubation of Fluosol with plasma led to C3 conversion, decrement in CH50, and generation of C5a-related PMN aggregating activity; EDTA prevented such activation, while EGTA did not, suggesting that it proceeded via the alternative C pathway. Infusion of Fluosol into rabbits produced hypoxemia, neutropenia, thrombocytopenia, and pulmonary leukostasis, mimicking abnormalities previously demonstrated in rabbits receiving infusions of zymosan-activated plasma C. These deleterious responses to Fluosol were diminished by premedicating rabbits with corticosteroids (which had seemed to benefit when used empirically in our patient). In vitro and in vivo, Fluosol's effects were reproduced by Pluronic F-68, the nonionic detergent used to maintain the emulsion stability of Fluosol-DA. We conclude that adverse reactions to Fluosol are probably mediated by C activation and that steroid premedication may prevent them in susceptible patients.

Rapid in vivo clearance of C5ades arg: a possible protective mechanism against complement-mediated tissue injury.

C5ades arg is an important mediator of the tissue injury associated with intravascular complement activation and exerts its effects by causing granulocyte aggregation, leukoembolism, and oxidative damage to endothelial cells. We have now investigated some of the mechanisms responsible for deactivation of this potent complement fragment by studying the clearance of C5ades arg from the plasma of New Zealand white rabbits in vivo. Quantitative aggregation of human granulocytes was used as a bioassay for rabbit C5ades arg. After a bolus intravenous infusion of activated autologous plasma complement, athe plasma C5ades arg content peaked within 60 sec and was cleared rapidly and exponentially, with a mean half-life of 3.0 +/- 0.6 (S.E.M.) min. In neutropenic animals the half-life of C5ades arg was prolonged by 57% +/- 22 and splenectomy, but not hepatectomy, also significantly lengthened the half-life by 48% +/- 18. The most striking delay in C5ades arg clearance was induced by inhibition of endogenous proteases with EACA and aprotinin, which prolonged the half-life by 87% +/- 4 and 94% +/- 12, respectively. These data demonstrate that like other complement fragments such as C3a, C5ades arg has a very brief functional half-life in experimental animals and that although other mechanisms may be operative, endogenous proteases, circulating granulocytes, and the spleen may be important in deactivating this biologically important complement fragment. Impairment of these and other clearance mechanisms could lead to prolonged and excessive complement activation and subsequent exaggerated tissue injury.

Effect of staphylococcal alpha-toxin on neutrophil migration and adhesiveness.

The effect of staphylococcal alpha-toxin on the chemotactic response of human polymorphonuclear leukocytes was studied by measuring the migration of alpha-toxin-treated cells either through membrane filters toward C5a or under agarose toward N-formyl-l-methionyl-l-phenylalanine. At doses of greater than or equal to 5 hemolytic units, alpha-toxin depressed chemotactic responsiveness in both best systems. Further studies revealed that alpha-toxin was also a potent granulocyte aggregant at doses similar to those necessary for depressed chemotactic capacity. It is proposed that the inhibitory effect of this membrane-active toxin on chemotactic function may be related to increased granulocyte adhesiveness and that the pathogenic properties of alpha-toxin may in part by explained by these effects.

Cholesterol and atheroma lipids activate complement and stimulate granulocytes. A possible mechanism for amplification of ischemic injury in atherosclerotic states.

Previous studies in experimental myocardial infarction have suggested that PMNs and plasma C might interact to intensify ischemic injury. From the finding of large amounts of activated C (specifically C5a) in the plasma of a patient with severe, ulcerating atherosclerosis and the cholesterol embolization syndrome, we postulated that crystalline cholesterol might activate C and thereby amplify infarctive tissue damage. On simple incubation, crystalline cholesterol--as well as lipids from atheromata--activated plasma C; such plasma then potently aggregated normal PMNs and provoked them to damage cultured endothelial cells in vitro. The active principle in cholesterol-incubated plasma was of molecular weight and antigenicity consistent with C5a (or C5a[desarginine]). Optimal activation required the presence of Ig and an intact classical C pathway. Exposure of plasma to crystalline cholesterol by ulceration of, or hemorrhage into, atherosclerotic plaques might therefore activate C in vivo, promoting further ischemic damage by causing leukostatic-leukoembolic compromise of small vessels downstream from the site of activation.

Corticosteroids block binding of chemotactic peptide to its receptor on granulocytes and cause disaggregation of granulocyte aggregates in vitro.

Inhibition of complement-mediated granulocyte aggregation has recently been proposed as a mechanism of action of high-dose corticosteroids in shock states. Postulating that such inhibition might be effected through alteration of receptors function, we examined the effect of methylprednisolone (MP), hydrocortisone (HC), and dexamethasone (DEX) on the extent and kinetics of binding of the synthetic chemotaxin f-methionine-leucine-phenylalanine (FMLP) to its specific receptor on the granulocyte surface. Dose-dependent inhibition of binding was observed at corticosteroid concentrations paralleling plasma levels achieved with 30 mg/kg intravenous bolus therapy; the order of potency was MP greater than HC greater than DEX. Receptor number was unaffected by steroid exposure, but the steroids effected a decrease in association rate constant for the FMLP-receptor interaction (35% of N for 0.2 mg/ml MP), leading to decreased receptor-ligand affinity. Dissociation kinetics, as examined by cold-chase experiments, were unaltered by the corticosteroids. Furthermore, in addition to the inhibition of aggregation previously reported, aggregated granulocytes were found to disaggregate upon addition of corticosteroids; the order of potency was again MP greater than HC greater than DEX, with an MP concentration of approximately 2-3 mg/ml required to effect complete disaggregation. We conclude that corticosteroids can displace FMLP from the granulocyte surface by slowing association while allowing dissociation to proceed; altered kinetics of receptor-FMLP interaction may explain both the inhibition of granulocyte aggregation and granulocyte disaggregation. If these observations also hold for physiologic stimuli (such as C5adesarginine, which behaves similarly with respect to aggregation, inhibition, and disaggregation), such kinetic changes may be important in the clinical effects of very high-dose corticosteroids such as are administered in shock.

Deleterious effects of endotoxin on cultured endothelial cells: an in vitro model of vascular injury.

The effects of endotoxin-triggered granulocytes on the viability of endothelial cells in vitro was investigated. Endotoxin or its lipid A component caused granulocytes to adhere to and significantly damage cultured endothelial cells. Fresh serum is not necessary but does amplify both adherence and endothelial injury. Much of the endothelial injury was inhibited by free-radical scavengers or by blocking granulocyte adhesion to endothelial cells and appears to result from free radical production by the stimulated granulocyte. Studies in this model suggest a pathogenic role for the endotoxin-triggered granulocyte in the Shwartzman reaction and perhaps related clinical disorders.

Reversal of hemodialysis granulocytopenia and pulmonary leukostasis: A clinical manifestation of selective down-regulation of granulocyte responses to C5adesarg.

The transient granulocytopenia of hemodialysis results indirectly from plasma complement activation by dialyzer cellophane membranes. The C5a(desarg) so produced can induce reversible granulocyte aggregation in vitro and in vivo, and we hypothesized that the pulmonary leukostasis responsible for the granulocytopenia results from embolization of aggregates formed under the influence of C5a(desarg) produced in the dialyzer. These studies were designed to measure C5a(desarg) generation during dialysis by granulocyte aggregometry and to determine the reason for the transience of the leukostasis. C5a(desarg) generation was equally evident throughout dialysis, persisting well after granulocytopenia had reversed, and dialyzer-induced complement activation was insufficient to produce significant depletion of plasma complement titers. That granulocyte deactivation might be responsible for the transience was suggested by the absence of the usual granulocytopenia in a patient with uniquely high levels of C5a(desarg) in his predialysis plasma. Granulocytes drawn from seven stable uremic patients after granulocytopenia had reversed exhibited a dose-related, selective and irreversible refractoriness to stimulation with C5a(desarg), but their responses to n-formyl-Met-Leu-Phe remained normal. Identical deactivation was produced in normal cells by short- or long-term exposure of C5a(desarg) in vitro. These studies suggest that C5a(desarg) is indeed generated by the dialyzer throughout hemodialysis and that the transience of the leukostasis and granulocytopenia is due to selective down-regulation of cellular responses to C5a(desarg)-a phenomenon that hitherto has been described only in vitro and that may be important in limiting the deleterious effects of adherent granulocytes on the endothelium in patients with intravascular complement activation.

Complement activation and neutropenia occurring during cardiopulmonary bypass.

Complement activation and pulmonary leukostasis with neutropenia occur in hemodialysis and filtration leukapheresis, with attendant pulmonary dysfunction. Wondering whether similar phenomena might attend cardiopulmonary bypass (CPB), we studied 34 patients undergoing coronary artery bypass operations. As in the other extracorporeal circulation systems, neutropenia (mean 44.7% +/- 4.3% SEM of prebypass PMN count) occurred during the first half hour of bypass and then a rebound neutrophilia followed. CH50 and C3H50 fell 22% to 25% (p for CH50 less than 0.01) during bypass, but C3 conversion and C5a were not demonstrable in patient plasmas. Nonetheless, polymorphonuclear neutrophils (PMNs) harvested late in bypass showed low adherence to nylon and selective chemotactic and aggregative insensitivity to C5a--functional aberrations which are seen after exposure to activated complement. Furthermore, smaller infusions of activated complement into animals produced neutropenia than were required to achieve a detectable [C5a] in the plasma. We conclude that neutropenia during CPB probably results from complement activation below the threshold of detection; complement-stimulated PMNs deserve study as possible mediators of tissue injury occurring during CPB.

Complement-induced granulocyte aggregation in vivo.

Previous studies from our laboratories have demonstrated that granulocytes (PMNs), when exposed to activated complement (C) (specifically C5a), will aggregate and be provoked to damage cultured endothelial cells in vitro; it was postulated that these phenomena might also occur in vivo, constituting a previously unsuspected mechanism of immune tissue damage. The studies here presented confirm by intravital microscopy that PMN aggregation and leukoembolization in fact occur in live animals when C is activated or C5a is infused, and that these are accompanied by extravasation of plasma proteins in a pattern suggesting endothelial damage. It is concluded that altered microvascular behavior of PMNs is a possible pathogenetic mechanism in disease states associated with C activation.

Sudden blindness in acute pancreatitis. Possible role of complement-induced retinal leukoembolization.

During an episode of acute alcoholic pancreatitis, severe visual loss and the funduscopic appearance of Purtscher's retinopathy-a syndrome thought to be caused by posterior retinal microembolization-developed in a patient. We propose that emboli in this case may have consisted of aggregated granulocytes since plasma samples from eight to 12 patients with subsequently studied acute pancreatitis caused granulocyte aggregation in vitro. The aggregant was demonstrated to be an activated fragment of the complement system, derived from C5. Since we could generate identical granulocyte aggregating activity by treating serum or purified C5 with trypsin, we suggest that proteases released from an inflamed pancreas might have produced a C5-derived aggregant in this case, as well as in three other previously reported cases of acute pancreatitis and Purtscher's retinopathy. We conclude that complement-induced leukoembolization may be a previously unsuspected cause of vital-tissue damage.