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.

Corticosteroids inhibit complement-induced granulocyte aggregation. A possible mechanism for their efficacy in shock states.

Granulocyte (PMN) aggregation and embolization may underlie complement (C)-mediated organ dysfunction in such syndromes as hemodialysis neutropenia and Purtscher's ischem;c retinopathy. Because of clinical and pathologic parallels, we have further suggested a role for this phenomenon in the genesis of the adult respiratory distress syndrome (ARDS). Because corticosteroids are commonly used in immune diseases, and have particularly been claimed efficacious in shock and ARDS, we tested the capability of methylprednisolone (MP), hydrocortisone (HC), and dexamethasone (DEX) to inhibit PMN aggregation. Aggregation engendered in vitro by zymosan-activated plasma (ZAP) was inhibited by MP and HC at concentrations approximating plasma levels achieved with the large bolus (30 mg/kg i.v) therapy advocated in shock states; DEX was almost without effect. Using intravital fluorescence microscopy, we observed PMN aggregation and embolization in the mesenteric vessels of rats given intra-arterial infusions of ZAP; this was also prevented by pretreatment with 30 mg/kg MP. Steroid inhibition of aggregation seemed not to involve disruption of receptor function, because aggregation induced by alternative agents, n-formyl-Met-Leu-Phe and the ionophore A23187, was also inhibited by MP. Moreover, corticosteroid inhibition of PMN prostaglandin synthesis is also an unlikely explanation for our results, since aspirin and ibuprofen failed to block aggregation and arachidonic acid neither effected aggregation itself nor ameliorated the steroid effect. Our studies provide a plausible rationale for the empiric observation that high-dose corticosteroids may benefit patients with syndromes associated with microvascular leukostasis.

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.

Hemodialysis leukopenia. Pulmonary vascular leukostasis resulting from complement activation by dialyzer cellophane membranes.

Acute leukopenia occurs in all patients during the first hour of hemodialysis with cellophanemembrane equipment. This transient cytopenia specifically involves granulocytes and monocytes, cells which share plasma membrane reactivity towards activated complement components. The present studies document that complement is activated during exposure of plasma to dialyzer cellophane, and that upon reinfusion of this plasma into the venous circulation, granulocyte and monocyte entrapment in the pulmonary vasculature is induced. During early dialysis, conversion of both C3 and factor B can be demonstrated in plasma as it leaves the dialyzer. Moreover, simple incubation of human plasma with dialyzer cellophane causes conversion of C3 and factor B, accompanied by depletion of total hemolytic complement and C3 but sparing of hemolytic C1. Reinfusion of autologous, cellophane-incubated plasma into rabbits produces selective granulocytopenia and monocytopenia identical to that seen in dialyzed patients. Lungs from such animals reveal striking pulmonary vessel engorgement with granulocytes. The activated complement component(s) responsible for leukostasis has an approximate molecular weight of 7,000-20,000 daltons. Since it is generated in C2-deficient plasma and is associated with factor B conversion, it is suggested that activation of complement by dialysis is predominantly through the altermative pathway.

Complement (C5-a)-induced granulocyte aggregation in vitro. A possible mechanism of complement-mediated leukostasis and leukopenia.

Activated plasma complement will induce biphasic aggregation of human granulocytes dectable by standard nephelometric techniques. The responsible active component was suggested to be C5a by molecular weight and heat-stability assays; moreover, aggragating activity was ablated by anti-C5 but not anti-C3 antibodies. C5a prepared by trypsinization of purified C5 reproduced the aggregating activity of whole activated plasma, whereas plasma from a C5-deficient donor did not support aggregation. Embolization of granulocyte aggregates might be a previously unsuspected cause of leukostasis and pulmonary damage in various clinical situations where intravascular complement activation occurs.

Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. An in vitro model of immune vascular damage.

During hemodialysis, alternative pathway complement activation leads to pulmonary sequestration of granulocytes, with loss of pulmonary vascular endothelial integrity and, at times, protein-rich pulmonary edema. An in vitro model of this phenomenon was constructed utilizing 51Cr-labeled human umbilical vein endothelial cell cultures. In this system, granulocytes, when exposed to activated complement (C), induce endothelial damage; this injury is mediated primarily by oxygen radicals produced by the granulocytes. C5a appears to be the C component responsible for granulocyte-induced cytotoxicity; studies with cytochalasin B-treated granulocytes suggest that close approximation of the granulocytes and endothelial cells is necessary for maximal cell injury.

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.

Granulocyte aggregation as a manifestation of membrane interactions with complement: possible role in leukocyte margination, microvascular occlusion, and endothelial damage.

Activation products of the terminal complement cascade potently affect granulocyte function, inducing, for example, their migration toward (chemotaxis), and adherence to (opsonization), microbes, and stimulating their production of microbicidal oxygen radicals such as superoxide anion, and the like. We present studies that demonstrate that a C5-derived peptide, probably C5a, is a potent promoter of granulocyte and monocyte adhesion to endothelium (margination) and, in addition, causes granulocyte autoaggregation in vitro and in vivo. Although possibly beneficial by producing phagocyte clumps to mechanically entrap unwanted microbes, such aggregates may be deleterious, particularly if sustained, especially in the lung.

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.

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.

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.

Digital integration of granulocyte aggregation responses. A simple and reproducible method for the quantitation of granulocyte adhesiveness.

The pulmonary leukostasis and lung dysfunction associated with intravascular complement activation results from C5a-mediated granulocyte (GR) aggregation, a phenomenon which can be reproduced in vitro using standard nephelometric techniques. To produce a more subjective measure of the extent and rate of GR aggregation responses we added a digital integrator to the system. The validity of this approach was substantiated by the close correlation between the aggregating and chemotactic activities of C5a and N-formyl-methionine-leucine-phenylalanine. Use of this technique enabled us to define the dose-response relationship of the aggregation produced by the cationophore A23187 and the inhibitory effect of tetracaine on divalent cation-dependent aggregation responses. The aggregation produced by these three stimuli does not result primarily from simple cross-linking of surface changes because, unlike the passive cell-cell association produced by the cation poly-L-lysine, it is not inhibited by anionic poly-L-glutamic acid. The importance of microtubules as regulators of GR adhesiveness was substantiated by the inhibitory effects of colchicine (but not lumicolchicine) on aggregation in this system. These data suggest that this integration of light transmission increments is a useful adjunct to this basic technique, whether used as a bioassay for chemotactic stimuli or as a model to study the many factors which regulate GR adhesiveness.

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.

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.

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.

Acquired granulocyte abnormality during drug allergic reactions: possible role of complement activation.

A profound defect in granulocyte chemotaxis was documented in an otherwise healthy 21-yr-old man who failed to localize granulocytes to an area of cellulitis during an allergic reaction to cephalothin. During the period of drug allergy, characterized by urticaria, eosinophilia, and profound hypocomplementemia, in vitro migration of the patient's granulocytes in the Boyden chamber was markedly impaired. Although devoid of hemolytic complement activity, the patient's serum possessed supranormal chemotactic activity, even following heat inactivation, suggesting the presence of chemotactically active complement split products. Chemotactic function improved concomitantly with steroid therapy and normalization of serum complement levels, and was entirely normal following clinical recovery and cessation of steroid therapy. The chemotactic abnormality noted in the patient's cells was reproduced in normal granulocytes by preincubation either with patient serum or with cobra venom-activated fresh (but not heated) normal serum, suggesting that in vivo exposure of granulocytes to activated complement was responsible for the patient's abnormal chemotactic response. This mechanism may contribute to the increased infection propensity noted in other conditions characterized by in vivo complement activation, such as rheumatoid arthritis and systemic lupus erythematosis.

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.

An adverse pulmonary reaction to cryoprecipitate in a hemophiliac.

An adult with classif Hemophilia A experienced a very severe reaction to transfusion with cryoprecipitate which was manifested as an adverse pulmonary reaction with marked hypoxemia in spite of oxygen therapy. The patient had neither leukoagglutinins nor lymphocytoxic, anti-platelet, or anti-Gm antibodies. His IgA level was normal. The possibility that debris in the cryoprecipitate from leukocytes and platelets contributed to the reaction is discussed.

Complement-mediated granulocyte dysfunction in paroxysmal nocturnal hemoglobinuria.

In paroxysmal nocturnal hemoglobinuria (PNH), infection, both viral and bacterial, disproportionate to the mild neutropenia seen in many such patients is responsible for significant morbidity. We report impaired granulocyte chemotaxis efficiency which may contribute to the problems induced by bacterial infections. PNH (but not normal) granulocytes, after exposure to very small concentrations of activated serum complement components, migrate poorly, as documented by their inhibited chemotaxis toward bacterial products or activated complement components in Boyden chambers. The granulocytes remain intact, excluding trypan blue, phagocytosing, and killing bacteria, despite this activated complement exposure. It is also suggested that this chemotactic defect may involve only a clone of cells, analogous to the clonal lysis of PNH erythrocytes; those few granulocytes capable of migration after exposure to activated complement contain normal quantities of leukocyte alkaline phosphatase (LAP), in contrast to the LAP deficiency of the overall PNH granulocyte population. Since bacterial infection may initiate or potentiate hemolysis, one of the major symptoms of the disease, these results could explain much of the morbidity of PNH.