Harry S. Jacob

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.

Complement and Leukocyte-Mediated Pulmonary Dysfunction in Hemodialysis

During hemodialysis, cardiopulmonary decompensation may appear in uremic patients, possibly caused by plugging of pulmonary vessels by leukocytes. In 34 patients we noted leukopenia (20% of initial levels) during hemodialysis that in 15 was associated with impaired pulmonary function. When we infused autologous plasma, incubated with dialyzer cellophane, into rabbits and sheep, sudden leukopenia and hypoxia occurred, with doubling of pulmonary-artery pressures and quintupling of pulmonary-lymph effluent. Histologic examination showed severe pulmonary-vessel-leukostasis and interstitial edema. The syndrome was prevented by preinactivation of complement but was reproduced by infusions of plasma in which complement was activated by zymosan. Thus, acute pulmonary dysfunction from complement-mediated leukostasis may play a major part in the acute cardiopulmonary complications of cellophane-membrane hemodialysis.

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.

Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.

Heme proteins such as myoglobin or hemoglobin, when released into the extracellular space, can instigate tissue toxicity. Myoglobin is directly implicated in the pathogenesis of renal failure in rhabdomyolysis. In the glycerol model of this syndrome, we demonstrate that the kidney responds to such inordinate amounts of heme proteins by inducing the heme-degradative enzyme, heme oxygenase, as well as increasing the synthesis of ferritin, the major cellular repository for iron. Prior recruitment of this response with a single preinfusion of hemoglobin prevents kidney failure and drastically reduces mortality (from 100% to 14%). Conversely, ablating this response with a competitive inhibitor of heme oxygenase exacerbates kidney dysfunction. We provide the first in vivo evidence that induction of heme oxygenase coupled to ferritin synthesis is a rapid, protective antioxidant response. Our findings suggest a therapeutic strategy for populations at a high risk for rhabdomyolysis.

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.

Complement-induced granulocyte aggregation: an unsuspected mechanism of disease.

The capacity of blood cells to aggregate, best exemplified by the response of platelets to vascular injury, is generally thought to be beneficial. However, if aggregation occurs inappropriately—tha...

Association of complement activation and elevated plasma-C5a with adult respiratory distress syndrome. Pathophysiological relevance and possible prognostic value.

Clinical and experimental observations suggest that aggregation of polymorphonuclear granulocytes (PMN) in response to activated complement (C) might contribute to the genesis of the adult respiratory distress syndrome (ARDS), aggregating PMN causing pulmonary dysfunction by becoming lodged in the lung as leucoemboli. PMN-aggregating activity can be detected in C-activated plasma and reflects C5a levels. In 61 patients at risk for ARDS a strong and highly significant correlation was found between the presence of PMN-aggregating activity in the plasma and the development of ARDS; this correlation was also significant when patients with sepsis were excluded from analysis. In patients followed prospectively detection of elevated C5a seemed to be a useful predictor of ARDS. Since corticosteroids have been shown to inhibit PMN aggregation both in vitro and in vivo, the evidence for a role for PMN aggregation in the genesis of ARDS supports the use of corticosteroids in this disorder.

Abnormal Adherence of Sickle Erythrocytes to Cultured Vascular Endothelium: POSSIBLE MECHANISM FOR MICROVASCULAR OCCLUSION IN SICKLE CELL DISEASE

The abnormal shape and poor deformability of the sickled erythrocyte (RBC) have generally been held responsible for the microvascular occlusions of sickle cell disease. However, there is no correlation between the clinical severity of this disease and the presence of sickled RBC. In searching for additional factors that might contribute to the pathophysiology of sickle cell disease, we have investigated the possibility that sickle RBC might be less than normally repulsive of the vascular endothelium. After RBC suspensions are allowed to settle onto plates of cultured human endothelial cells, normal RBC are completely removed by as few as six washes. In contrast, sickle RBC remain adherent despite multiple washes. On subconfluent culture plates, normal RBC are distributed randomly, whereas sickle RBC cluster around endothelial cells. Sickle RBC adherence is not enhanced by deoxygenation but does increase with increasing RBC density. The enzymatic removal of membrane sialic acid greatly diminishes the adherence of sickle RBC to endothelial cells, suggesting that sialic acid participates in this abnormal cell-cell interaction. Although net negative charge appears normal, sickle RBC mainfest an abnormal clumping of negative surface charge as demonstrated by localization of cationized ferritin. These abnormalities are reproduced in normal RBC loaded with nonechinocytogenic amounts of calcium. We conclude that sickle RBC adhere to vascular endothelial cells in vitro, perhaps caused by a calcium-induced aberration of membrane topography. This adherence may be a pathogenetic factor in the microvascular occlusions characteristic of sickle cell disease.

Hemin: a possible physiological mediator of low density lipoprotein oxidation and endothelial injury.

Oxidized low density lipoprotein (LDL), formed in vivo from presently unknown reactions, may play a role in atherogenesis. In vitro, transition metals such as iron and copper will facilitate LDL oxidation, but these metals are unlikely to exist in free form in normal body fluids. We have explored the possibility that LDL oxidation may be promoted by heme, a physiologically ubiquitous, hydrophobic, iron-containing compound. Indeed, during several-hour incubation, heme caused extensive oxidative modification of LDL; however, such modification requires only minutes in the presence of small amounts of H2O2 or preformed lipid hydroperoxides within the LDL. Oxidative interactions between heme, LDL, and peroxides lead to degradation of the heme ring and consequent release of heme iron, which further accelerates heme degradation. Coupled (evidently iron-catalyzed) heme degradation and LDL oxidation are both effectively inhibited by hydrophobic antioxidants and iron chelators. That such hemin-induced LDL oxidation may be involved in atherogenesis is supported by the finding that LDL oxidized by hemin is extremely cytotoxic to cultured aortic endothelial cells. Overall, these investigations not only lend support to the idea that LDL oxidation by physiological substances such as heme may play a role in the process of atherogenesis but also may have broader implications, as similar oxidative reactions between heme and unsaturated fatty acids may occur consequent to hemorrhagic injury.

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 Purtschers 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.