Infusion Of Cobra Venom Factor Research Articles

Elevation of rat plasma P-selectin in acute lung injury

Acute lung injury in the rat caused by intravenous (i.v.) infusion of cobra venom factor (CVF) or lipopolysaccharide (LPS) is mediated by P-selectin-dependent neutrophil infiltration into the lung. In these lung injury models, P-selectin expression is induced on lung vascular endothelial cells after the CVF or LPS infusion, suggesting soluble P-selectin derived from inflamed sites might also be elevated. Here we established a sensitive enzyme-linked immunosorbent assay (ELISA) to measure soluble P-selectin in plasma, a potential marker of lung injury. Nine anti-rat P-selectin monoclonal antibodies that we established previously were first classified into 5 groups based on real-time biospecific interaction analyses, and used to develop a sandwich ELISA for accurately measuring the amount of soluble P-selectin in plasma. We then used this ELISA to measure the plasma P-selectin levels in Long Evans, Wistar, and Sprague–Dawley rats after the i.v. infusion of CVF or LPS. The elevation in P-selectin levels was significantly different among the strains, but it consistently correlated with the extent of lung inflammation, measured by myeloperoxidase levels in the lung tissues. Thus, our results indicate that the soluble P-selectin in plasma could serve as a sensitive biomarker reflecting lung inflammation, which is of clinical importance for detecting and preventing severe lung injury.

Complement inhibitors selectively attenuate injury following administration of cobra venom factor to rats

Systemic activation of complement is a pathophysiological response common to severe disturbances such as hemorrhagic shock, major burn injury and sepsis. Intravenous infusion of cobra venom factor (CVF) has been used as an animal model of acute respiratory distress syndrome (ARDS), and reliably and selectively induces rapid intravascular activation of the complement system, leading to acute organ damage. In the present study, we have used different complement inhibitors to investigate the roles of complement products in CVF-induced responses in the rat. Rats were treated with either a C5a receptor antagonist (C5aRA, AcF-[OP( d-Cha)WR], 1 mg/kg i.v. or 10 mg/kg p.o.), a C3a receptor antagonist (C3aRA, N(2)-[(2,2-diphenylethoxy)acetyl]- l-arginine, 0.1 mg/kg i.v.) or a convertase inhibitor, rosmarinic acid (RMA, 10 mg/kg i.v.), prior to CVF-induced complement challenge. Intravenous CVF resulted in hallmark events evident in the development of ARDS, including systemic neutropenia followed by neutrophil migration to the lung and bronchoalveolar vascular leakage, blood pressure alterations, and an increase in TNFα levels in both serum and bronchoalveolar lavage fluid. These hemodynamic changes were differentially inhibited by antagonism of C5a receptors, C3a receptors or by inhibition of the entire complement cascade using RMA. This evidence strongly implicates complement factors in the development of lung injury associated with systemic complement activation and identifies complement inhibition as a potential therapeutic target for acute syndromes such as ARDS and other severe systemic shock states mediated by activation of complement.

Pre-treatment of donor with 1-deamino-8-d-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient☆

Unlike cardiac or renal xenotransplants, the depletion of complement using cobra venom factor (CVF) does not improve pulmonary xenograft survival. Several cases suggest that the swine von Willebrand factor (vWF) may play a major role in presenting a different pathogenesis of pulmonary xenograft dysfunction from other organs. To evaluate the role of vWF and the complement system in mediating hyperacute vascular injury of pulmonary xenografts and elucidate pathogenesis of the injury, we performed swine-to-canine orthotropic single lung xenotransplantation after pre-treatment of 1-deamino-8-d-arginine vasopressin (DDAVP) and CVF. We set up three groups for lung xenotransplantation: group I served as the control group; group II, recipients pre-treated with CVF; group III, donors pre-treated with DDAVP (9 mg/kg, 3 days)/recipients pre-treated with CVF (60 u/kg). Hemodynamic data, coagulation and complement system parameters, and grafted lung pathologies were examined serially for 3h after transplantation. DDAVP infusion reduced the vWF content in swine lung tissue in vivo (7.7+/-2.4 AU/mg vs 16.0+/-5.6 AU/mg, P < 0.0001). Infusion of CVF 24 h prior to transplantation effectively depleted the recipient's serum C3 and complement hemolytic activity below the detectable range. Regardless of the use of CVF, both groups I and II transplanted with unmodified grafts showed an immediate drop in leukocytes and platelet counts after transplantation. However, in group III, in recipients transplanted with DDAVP pre-treated swine lung, the platelet count did not decrease after transplantation (P = 0.0295). The decrease of plasma antithrombin and fibrinogen tended to be attenuated in group III. Light microscopic examination revealed extensive vascular thromboses in both capillary and larger vessels, as well as early pulmonary parenchymal damage in groups I and II, but were rarely observed in group III. Complement inhibition alone was not enough to alleviate intravascular thrombosis, the main pathology in pulmonary xenotransplantation. Pre-infusion of DDAVP to the donor animal was effective in preventing platelet sequestration and attenuated intravascular thrombosis. It is suggested that the strategies targeting vWF would be promising for successful pulmonary xenotransplantation.

Procalcitonin release patterns in a baboon model of trauma and sepsis: Relationship to cytokines and neopterin

Procalcitonin (PCT) has been described as an early, discriminating marker of bacteria-associated sepsis in patients. However, little is known of its source and actions, in part because no appropriate animal models have been available. We tested the hypothesis that plasma PCT increases during various pathophysiological conditions, such as hemorrhagic shock and sepsis, which differ with regard to the degree of associated endotoxemia. We further hypothesized that in sepsis, PCT would be significantly different in survivors vs. nonsurvivors. Prospective, blinded analysis of previously collected plasma of experimental animals. Independent nonprofit research laboratory in a trauma hospital and a contract research institute. A total of 22 male baboons (17.5-31 kg). Hemorrhagic-traumatic shock was induced by hemorrhage for up to 3 hrs, reperfusion with shed blood and infusion of cobra venom factor (n = 7). By using a similar experimental setup, severe hyperdynamic sepsis was induced (n = 15) by intravenous infusion of live Escherichia coli (2 x 10(9) colony-forming units/kg) over 2 hrs, followed by antibiotic therapy (gentamicin 4 mg/kg twice a day). Plasma PCT at baseline was barely detectable, but levels increased significantly (p < .05) to 2+/-1.8 pg/mL 2 hrs after the start of reperfusion in the shock group, and to 987+/-230 pg/mL at 4 hrs after E. coli in the sepsis group. Levels were maximal between 6 and 32 hrs and had returned nearly to baseline levels at 72 hrs. Interleukin-6 levels paralleled the course of PCT measurements, whereas a significant increase in neopterin was seen at 24 hrs. PCT levels were approximately three times higher in the sepsis group than in the shock group, corresponding to endotoxin levels (at the end of hemorrhage, 286+/-144 pg/mL vs. 3576+/-979 pg/mL at the end of E. coli infusion; p = .003). PCT levels were significantly different at 24 hrs between survivors (2360+/-620 pg/mL) and nonsurvivors (4776+/-563 pg/mL) in the sepsis group (p = .032), as were interleukin-6 (1562+/-267 vs. 4903+/-608 pg/mL; p = .01) and neopterin/creatinine ratio (0.400+/-0.038 vs. 0.508+/-0.037; p = .032). PCT is detectable in the baboon as in humans, both in hemorrhagic shock and sepsis. PCT levels are significantly higher in sepsis than in hemorrhage, a finding that is probably related to the differences in endotoxin. The baboon can be used for the study of PCT kinetics in both models; PCT kinetics are clearly different from other markers of sepsis, either IL-6 or neopterin, in both models. There are significant differences between survivors and nonsurvivors in the sepsis model.

Leukocyte Sequestration in Pulmonary Microvessels and Lung Injury following Systemic Complement Activation in Rabbits

Inflammatory reactions are associated with sequestration of leukocytes in the lung. Complement activation leads to accumulation of leukocytes in alveolar septa and alveoli, to lung edema and hemorrhage. Although in organs other than the lung leukocytes interact with the vascular endothelium only in postcapillary venules, alveolar capillaries are considered to be the site of leukocyte sequestration in the lung. However, pulmonary venules and arterioles have not been investigated systematically after complement activation so far. A closed thoracic window was implanted in anesthetized rabbits; leukocytes and red blood cells were stained, and the movement of these cells was measured in superficial pulmonary arterioles, venules and alveolar capillaries using fluorescence video microscopy before and 30 and 60 min after infusion of cobra venom factor (CVF). Erythrocyte velocity and macrohemodynamic conditions did not change after CVF infusion and were not different from the sham-treated controls. The number of sticking leukocytes increased significantly compared to baseline and control: by 150% in arterioles and in venules and by 740% in alveolar capillaries within 60 min after CVF infusion. The width of alveolar septa in vivo was significantly enlarged after CVF infusion, indicating interstitial pulmonary edema. At the end of the experiments, myeloperoxidase activity was higher in the CVF group, showing leukocyte sequestration in the whole organ. It is concluded that complement activation by CVF induces leukocyte sequestration in lung arterioles, venules and alveolar capillaries and leads to mild lung injury.

C5a-dependent up-regulation in vivo of lung vascular P-selectin.

Acute lung injury in rats following systemic activation of complement by i.v. infusion of cobra venom factor (CVF) is known to be P-selectin dependent. In the current studies, infusion of CVF caused the appearance in plasma of C5a (as revealed by ELISA analysis) together with neutrophil chemotactic activity, which was totally blocked by addition in vitro of anti-rat C5a. Using a detector 125I-labeled antibody to rat P-selectin, we have demonstrated quantitative up-regulation in vivo of lung vascular P-selectin in a time-dependent manner after infusion of CVF. This up-regulation was almost completely blocked by prior complement depletion or by the infusion of anti-rat C5a. Platelet depletion did not affect CVF-induced up-regulation of lung vascular P-selectin, indicating that platelets were not the source of P-selectin. These results demonstrate that complement and, specifically, C5a are necessary for up-regulation of lung vascular P-selectin after systemic activation of complement.

Neutrophil chemotactic activity and C5a following systemic activation of complement in rats.

Using ELISA analysis, rat C5a was stimulated in serum from rats undergoing systemic activation of complement after intravenous infusion of purified cobra venom factor (CVF). Biological (neutrophil chemotactic) activity was also assessed. Serum levels of C5a were directly proportional to the amount of CVF infused. C5a and neutrophil chemotactic activity, peaked by 5 min, then plateaued. In vitro addition of anti-C5a to serum samples of CVF-infused rats totally abolished chemotactic activity, indicating that all biological activity could be ascribed to C5a. Blood neutrophils obtained from CVF-infused animals showed a significant upregulation of CD11b, the increase being reduced (38%) in animals pretreated with anti-C5a. These findings indicate that infusion of CVF into rats produces generation of C5a, all chemotactic activity in serum being related to C5a. The in vivo generation of C5a is, at least inpart, responsible for upregulation of CD11b on blood neutrophils.

Requirement and role of C5a in acute lung inflammatory injury in rats.

The complement activation product, C5a, may play a key role in the acute inflammatory response. Polyclonal antibody to rat C5a was used to define the requirements for C5a in neutrophil-dependent inflammatory lung injury after systemic activation of complement by cobra venom factor (CVF) or after intrapulmonary deposition of IgG immune complexes. In the CVF model, intravenous infusion (but not intratracheal instillation) of anti-C5a produced a dose-dependent reduction in lung permeability and in lung content of myeloperoxidase. In C6-deficient rats, CVF infusion caused the same level of lung injury (measured by leak of 125I-albumin) as found in C6-sufficient rats. In the IgG immune complex model of lung injury, anti-C5a administered intratracheally (but not intravenously) reduced in a dose-dependent manner both the increase in lung vascular permeability as well as the buildup of lung myeloperoxidase. Treatment with anti-C5a greatly suppressed upregulation of lung vascular intercellular adhesion molecule-1 (ICAM-1). This was correlated with a substantial drop in levels of TNFalpha in bronchoalveolar fluids. These data demonstrate the requirement for C5a in the two models of injury. In the IgG immune complex model, C5a is required for the full production of TNFalpha and the corresponding upregulation of lung vascular ICAM-1.

Measurement of neutrophil content in brain and lung tissue by a modified myeloperoxidase assay.

Myeloperoxidase (MPO) activity is assessed for the quantification of neutrophil accumulation in tissues. In particular, it may be used to support in vivo data on leukocyte kinetics obtained by intravital microscopy and to clarify whether phenomena observed on the organ surface reflect the situation of the whole organ microcirculation. Previous measurements of MPO activity were limited by interference with other peroxidases and by inhibition of MPO activity by specific enzymes. To circumvent these limitations, a modified assay was devised that combined a two-step tissue homogenization technique with heat incubation in a continuous photometric measurement. MPO activity was quantified in neutrophils isolated from rat and rabbit whole blood, rat brain and rabbit lung and compared with intravital microscopic data on leukocyte accumulation. The modified assay is characterized by high reproducibility, strong correlation of MPO activity with number of neutrophils and full recovery of neutrophils added to tissue homogenate. MPO activity per neutrophil was 342.9 +/- 11.7 mU/10(6) cells in rats and 40.3 +/- 0.8 mU/10(6) cells in rabbits. MPO activity in tissue was significantly lower in rat brains (18.9 +/- 29.7 mU/g) as compared to rabbit lungs (741 +/- 67 mU/g). Whereas global cerebral ischemia/reperfusion did not increase MPO activity in rat brain (18.1 +/- 26.1 mU/g), intravenous infusion of cobra venom factor (1,447 +/- 407 mU/g) or endotoxin (1,439 +/- 285 mU/g), enhanced MPO activity in rabbit lung. These results parallel microcirculatory data from the organ surface. Therefore they supplement the intravital microscopic observations by demonstrating that these are indeed representative of deeper parenchymal tissue areas.

Requirements for L-selectin in neutrophil-mediated lung injury in rats.

L-selectin requirements in three models of acute lung injury in rats have been identified: systemic activation of complement after intravenous infusion of cobra venom factor (CVF) and intrapulmonary deposition of IgG or IgA immune complexes. In the CVF model of lung injury, treatment of rats with hamster monoclonal IgG anti-rat-L-selectin (HRL-1) induced significant neutropenia, necessitating the use of F(ab')2 fragments, which did not cause neutropenia. Treatment of rats with F(ab')2 anti-L-selectin (HRL-1) resulted in significant reductions in lung permeability and hemorrhage in the CVF model. Morphologically, this treatment abrogated adhesive interactions of neutrophils with the pulmonary vascular endothelium. In the IgG immune complex model of injury, the parameters of injury were significantly reduced as a result of treatment with HRL-1. In both models protection was associated with reductions in lung myeloperoxidase content. Treatment of rats with a F(ab')2 form of hamster monoclonal IgG non-blocking anti-L-rat selectin, HRL-2, failed to show protective effects in the CVF and IgG immune complex models of lung injury. In the IgA immune complex model of injury, which is neutrophil-independent and related to toxic products from pulmonary macrophages, no protective effects of anti-L-selectin (HRL-1) were found. Therefore, in neutrophil-dependent and oxygen radical mediated lung injury, L-selectin plays a requisite role in tissue recruitment of neutrophils. In the neutrophil-independent model of lung injury, no requirement for L-selectin appears to exist.

Protective effects of selectin chimeras in neutrophil-mediated lung injury.

Recombinant selectin chimeric molecules featuring the joining of the extracellular domains of L-, P-, and E-selectin to the CH2 and CH3 domains of human IgG1 have been evaluated for their ability to protect against neutrophil-dependent lung injury in rats after systemic activation of C caused by vascular infusion of cobra venom factor (CVF) or lung injury that follows intrapulmonary deposition of IgG immune complexes. Previous studies using anti-selectin antibodies have suggested that the former model is P-selectin dependent, whereas the latter is E-selectin dependent. Requirements for L-selectin have not been identified because of lack of reagents. For the current studies employing the CVF model of lung injury, infusion of P-selectin-Ig chimera reduced injury (as assessed by changes in permeability and hemorrhage) in a dose-dependent manner, with parallel reductions in lung myeloperoxidase (MPO) content. Similar results were obtained with the L-selectin-Ig chimera, whereas the E-selectin-Ig chimera was not protective and failed to alter MPO content. In contrast, in the IgG immune complex model of lung injury, the L- and E-selectin-Ig chimeras both showed dose-related protective effects and reductions in MPO content, whereas the P-selectin-Ig chimera failed to protect against injury and did not alter MPO content in this model of lung injury. In all cases of blocking of injury, this was incomplete, suggesting multi-selectin engagement or inadequate amounts of selectin-Ig chimeras employed. These data indicate that neutrophil recruitment and attendant lung injury in the CVF model are L- and P-selectin dependent and E-selectin-independent, whereas in the IgG immune complex model, neutrophil recruitment and lung injury are L- and E-selectin-dependent but independent of P-selectin. Thus, differing selectin requirements for acute inflammatory lung injury have been identified.

Protective effects of oligosaccharides in P-selectin-dependent lung injury.

Neutrophil recruitment into tissues is a multistep process involving sequential engagement of adhesion molecules, including selectins (E,P,L), which are reactive with oligosaccharides, and the family of beta 2 integrins which are reactive with endothelial intercellular adhesion molecules. These processes result in the initial rolling of leukocytes along the endothelial surfaces, followed by the firm attachment of leukocytes to the endothelium. The intravenous infusion of cobra venom factor into rats results in acute lung injury that is neutrophil-dependent, oxygen radical mediated and P-selectin-dependent. Here we report that infusion of sialyl-Lewis X, a ligand for P-selectin, dramatically reduced lung injury and diminished the tissue accumulation of neutrophils, whereas irrelevant oligosaccharides had no such effects. These results suggest that sialyl-Lewis X carbohydrates may be used as a new strategy for anti-inflammatory therapy.

Inhibition of lung inflammatory reactions in rats by an anti-human IL-8 antibody.

IL-8 belongs to the family of chemotactic cytokines and may play an important role in the inflammatory response. In the current studies, a murine mAb (DM/C7) to human rIL-8 was found to have protective effects in inflammatory lung injury in rats. DM/C7 was nonreactive with the rat cytokine-induced neutrophil chemoattractant peptide. In vivo, DM/C7 blocked the glycogen-induced accumulation of neutrophils in rats and was highly protective against lung and dermal vascular injury after deposition of IgG immune complexes. The latter model of injury has recently been shown to be E-selectin dependent. The protective effects of DM/C7 correlated with reduced tissue accumulation of neutrophils, as measured by myeloperoxidase content. DM/C7 reacted with an epitope expressed by TNF-alpha-stimulated rat pulmonary artery endothelial cells and with the pulmonary vascular endothelium after intrapulmonary deposition of IgG immune complexes. In the model of IgG immune complex-induced lung injury, the protective effects of DM/C7 were abolished by prior absorption of the antibody with human rIL-8. Polyclonal antibody to cytokine-induced neutrophil chemoattractant peptide failed to protect against IgG immune complex-induced vascular injury even though this antibody blocked the in vitro chemotactic activity of cytokine-induced neutrophil chemoattractant. In the model of rapidly developing lung injury due to systemic activation of C after infusion of cobra venom factor, DM/C7 was not protective. As well, in the neutrophil-independent model of IgA immune complex-induced lung injury, treatment with DM/C7 was not protective. These data indicate that in inflammatory lung injury that is linked to E-selectin-dependent recruitment of neutrophils in rats, antibody to human IL-8 also blocks recruitment of neutrophils and thereby affords protection against lung injury. The data suggest the presence of an IL-8-like product in this model of lung injury.

Role of leukocyte adhesion molecules in complement-induced lung injury.

The bolus i.v. infusion of cobra venom factor into rats results in acute lung injury that is neutrophil-dependent, oxygen radical-mediated, and requires CD18. In our studies a more precise definition regarding the role of beta-integrins and requirements for cytokines was obtained by the use of blocking antibodies. Lung injury was quantitated by changes in permeability (leakage of 125I-BSA) and hemorrhage (extravasation of 51Cr-RBC). In animals treated with anti-CD11a, the permeability and hemorrhage parameters were reduced by 30 and 29%, respectively. Treatment with anti-CD11b resulted in reductions in permeability and hemorrhage by 53 and 48%, respectively, whereas anti-intercellular adhesion molecule-1 reduced the parameters of injury by 60 and 75%, respectively. Not surprisingly, treatment with antibodies to very late Ag-4, TNF-alpha and IL-1 failed to show any protective effects, which contrasts to the requirements for these molecules in lung injury after deposition of IgG immune complexes. Protective interventions were associated with a reduction in lung content of myeloperoxidase. These studies indicate that, in the cobra venom factor model of acute lung injury in rats, engagement of Mac-1, lymphocyte function-associated Ag-1, and intercellular adhesion molecule-1 are essential, whereas, in contrast to other models of neutrophil mediated lung injury, cytokines (TNF-alpha and IL-1) and very late Ag-4 are not required for the full development of injury.

Neutrophil-dependent acute lung injury. Requirement for P-selectin (GMP-140).

Rapid translocation of P-selectin (GMP-140) from cytoplasmic granules to the cell membrane of endothelial cells promotes adhesive interactions with neutrophils which, when activated, damage the endothelium. The role of P-selectin in lung vascular endothelial injury in rats after systemic activation of complement by intravenous infusion of cobra venom factor has been assessed. Within 5-10 min after cobra venom factor infusion, the pulmonary vasculature demonstrated immunohistochemical expression of an epitope that reacts with anti-human P-selectin. Monoclonal antibody to human P-selectin blocked in vitro adherence of rat or human platelets (activated with thrombin) to neutrophils and was demonstrated to react with thrombin-activated rat platelets. The antibody did not react with rat neutrophils. In vivo, the antibody had strongly protective effects against cobra venom factor-induced pulmonary vascular injury as determined by permeability changes and hemorrhage. In parallel, lung myeloperoxidase content was greatly reduced and, by transmission electron microscopy, there was markedly diminished adherence of neutrophils to the pulmonary vascular endothelium and much diminished injury of endothelial cells, as defined by hemorrhage. These data indicate that anti-human P-selectin reacts with a pulmonary vascular antigen in rats and that this antigen is essential for the full expression of lung injury.

Roles of beta 2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury.

The roles of beta 2 integrin molecules in neutrophil accumulation and tissue injury have been examined by the use of antibodies that are reactive with human CD11b and CD18 and cross-react with the homologous epitopes on rat neutrophils. Adherence to rat pulmonary artery endothelial cells by human neutrophils and endothelial cell killing by phorbol ester-activated human neutrophils required CD11b, CD11c, and CD18. Companion adherence studies between rat neutrophils and endothelial cells revealed a requirement for both CD11b and CD18. Neither anti-CD11b nor anti-CD18 depressed in vitro responses (O2- generation and chemotactic migration) of rat neutrophils. The accumulation of neutrophils in glycogen-induced peritoneal exudates was diminished substantially in rats treated with either anti-CD18 or anti-CD11b. In oxidant-mediated acute lung injury induced by rapid intravascular infusion of cobra venom factor, treatment of rats with either anti-CD18 or anti-CD11b significantly attenuated injury as assessed by increases in vascular permeability and hemorrhage. These protective effects correlated morphologically with diminished adhesion of neutrophils to interstitial intrapulmonary capillary endothelial cells. In studies of immune complex (BSA-anti-BSA)-induced alveolitis and dermal vasculitis, anti-CD18 had protective effects at all doses of anti-BSA employed. The protective effects of anti-CD18 correlated with diminished neutrophil accumulation in tissues at lower doses of anti-BSA. Although anti-CD11b was not effective under the same experimental conditions, intratracheal administration of this antibody conveyed protection against immune complex-induced lung injury, suggesting that both CD11b and CD18 are required for the full expression of injury. The current studies also demonstrated that when surface-bound IgG immune complexes were treated with fresh rat serum, the increment in O2- and TNF alpha generated by alveolar macrophages was suppressed by anti-CD18, but not by anti-CD11b, suggesting a heretofore unrecognized role for CD18 in the O2- and TNF-alpha responses of alveolar macrophages. Thus, neutrophil beta 2 integrins play a requisite role for the full expression of complement-dependent and oxygen radical-mediated injury of the lung and dermal vasculature.

Effect of complement activation with cobra venom factor on pulmonary vascular permeability.

We examined the effect of acute complement activation on lung vascular permeability to proteins in awake sheep prepared with lung lymph fistulas. Complement was activated by cobra venom factor (CVF) infusion (400 U/kg for 1 h iv). Studies were made in two groups of sheep: 1) infusion of CVF containing the endogenous phospholipase A2 (PLA2) (n = 6); and 2) infusion of CVF pretreated with bromophenacyl bromide to inhibit PLA2 activity (n = 5). Intravascular complement activation transiently increased mean pulmonary arterial pressure (Ppa) and pulmonary vascular resistance (PVR) in both groups. Pulmonary lymph flow (Qlym) and lymph protein clearance (Qlym X lymph-to-plasma protein concentration ratio) were also transiently increased in both groups. Pulmonary vascular permeability to proteins was assessed by raising left atrial pressure and determining the lymph-to-plasma protein concentration ratio (L/P) at maximal Qlym. In both groups the L/P at maximal Qlym was not different from normal. In a separate group (n = 4), CVF-induced complement activation was associated with 111In-oxine granulocyte sequestration in the lungs. In vitro plasma from CVF-treated animals aggregated neutrophils but did not stimulate neutrophils to produce superoxide anion generation. Therefore, CVF-induced complement activation results in pulmonary neutrophil sequestration and in increases in PVR and lymph protein clearance. The increase in lymph protein clearance is due to increased pulmonary microvascular pressure and not increased vascular permeability to proteins.

Comparison of intravenous gamma globulin and a monoclonal anti-Fc receptor antibody as inhibitors of immune clearance in vivo in mice.

Fc-receptor-mediated clearance and nonspecific phagocytic clearance were assessed after the infusion of monomeric human IgG, heat-aggregated human IgG, and a monoclonal anti-mouse macrophage FcII receptor antibody (2.4G2) into normal mice. Each agent blocked Fc-receptor function in vivo, but 2.4G2 was much more potent per microgram than the other agents. Monomeric IgG in blocking doses did not affect other aspects of immune function. In contrast, aggregated IgG, and to a lesser extent, 2.4G2 reduced serum complement levels. In addition, these agents also caused moderate reductions in nonspecific phagocytic function. Monoclonal anti-mouse macrophage C3bi receptor antibody (Mac-1), another monoclonal antibody which binds to macrophage CR3 receptors without interfering with Fc-receptor function, also reduced serum complement and inhibited nonspecific phagocytic function. Complement depletion alone (produced by infusion of cobra venom factor) could not account for the observed changes in Fc receptor or nonspecific phagocytic function. We conclude that both monomeric IgG and anti-Fc-receptor antibodies can markedly inhibit Fc-receptor function in vivo; however, the pattern of physiologic changes produced by these agents differs.

Attenuation by 2,3-dihydroxybenzoic acid of acute lung injury induced by cobra venom factor in the rat.

Neutrophil-derived oxygen metabolites are thought to play an important role in the genesis of acute lung injury in a variety of diseases. In an effort to find an agent that might limit the injury, we evaluated the beneficial effects of 2,3-dihydroxybenzoic acid (DHB), a drug that has been safely administered to humans as an iron-chelating agent. Because experimental evidence has demonstrated that DHB can act as an inhibitor of free radical-induced reactions, we tested its protective effect against the neutrophil-dependent lung injury that occurs in rats after the intravenous infusion of cobra venom factor (CVF). Using a permeability index that measures the amount of intravenously administered 125I-albumin that accumulates in lung tissue, we found that pretreatment with DHB reduced (p less than 0.05) the lung injury in CVF-treated rats in a dose-dependent manner. Morphometric analysis of lung tissue indicated that the protection by DHB was not caused by inhibition of CVF-induced neutrophil sequestration within the lung vasculature. Because iron-saturated DHB did not attenuate lung injury, and because in vitro experiments demonstrated that DHB inhibited iron-hydrogen peroxide-induced peroxidation of phospholipid liposomes, we suspect that DHB may be protecting the lung via chelation of iron. We conclude that dihydroxybenzoic acid protects the rat lung from the neutrophil-dependent lung injury that occurs after cobra venom factor infusion. Because this drug has been safely administered to humans, it may have potential as an agent to prevent acute lung injury.

Additive effect of intravascular complement activation and brief episodes of hypoxia in producing increased permeability in the rabbit lung.

Systemic complement activation with intravascularly administered cobra venom factor (CVF) or infusion of either zymosan-activated rabbit plasma or a fifth component of complement fragment with anaphylatoxin activity in the rabbit have not caused significant increases in bronchoalveolar lavage albumin in rabbits (Webster, R. O., G. L. Larsen, B. C. Mitchell, A. J. Goins, and P. M. Henson. 1982. Am. Rev. Respir. Dis. 125:335-340). To assess if another stimulus (hypoxia) acting in concert with complement activation can produce significant lung injury, rabbits were challenged with CVF alone, 10 min of 12% oxygen alone, or CVF followed by a 10-min exposure to 12% oxygen. Either stimulus alone caused no significant changes in arterial oxygen, pulmonary resistance, or dynamic compliance during the 240 min of observation after either stimulus, and neither stimulus alone caused increased albumin accumulation in bronchoalveolar lavage over a 30-min period at the end of the experiment. However, the combination of insults significantly altered arterial oxygen, pulmonary resistance, and dynamic compliance while also increasing albumin and neutrophils recovered by lavage. The increase in lavage albumin did not appear to be due to hemodynamic events in that the pulmonary artery pressure increased acutely after CVF infusion and again during the hypoxic exposure, but was normal when albumin accumulation in the lung was measured. Neutrophil depletion with nitrogen mustard abolished all of these changes induced by CVF plus hypoxia. In addition, meclofenamate pretreatment and infusion during the 4-h study abolished the increases in lavage albumin and neutrophils as well as the increase in pulmonary artery pressure after CVF. Meclofenamate pretreatment did not, however, block accumulation of albumin in the lung (interstitium). We conclude that complement activation, as an isolated event, will not cause a significant increase in lavage albumin in this model. However, combining complement activation with an episode of hypoxia will lead to an increase in lavage albumin that is dependent on the presence of neutrophils for its expression. Meclofenamate treatment will prevent increases in lavage albumin and neutrophils while not preventing albumin accumulation in the lung (interstitium), suggesting a product of the cyclooxygenase pathway of arachidonic acid metabolism is needed to produce movement of albumin and/or neutrophils across the alveolar epithelium in this model.