Gerd O. Till

Intravascular activation of complement and acute lung injury. Dependency on neutrophils and toxic oxygen metabolites.

Intravascular activation of the complement system with cobra venom factor results in acute lung injury, which has been quantitated by increases in lung vascular permeability. Cobra venom factor preparations devoid of phospholipase A2 activity retain full lung-damaging capacity. The lung injury is associated with the preceding appearance of chemotactic activity in the serum coincident with the development of a profound neutropenia. The chemotactic activity is immunochemically related to human C5a. Morphologic studies have revealed discontinuities in the endothelial cell lining of lung alveolar capillaries, damage and/or destruction of endothelial cells in these areas, plugging of pulmonary capillaries with neutrophils that are in direct contact with vascular basement membrane, the presence of fibrin in alveolar spaces and in areas adjacent to damaged endothelial cells, and intraalveolar hemorrhage. Lung injury is dramatically attenuated in animals that have been previously neutrophil depleted. Teh intravenous injection of superoxide dismutase or catalase also provides significant protection from the pulmonary damage. Very little protection from the pulmonary damage. Very little protection is afforded by pretreatment of rats with antihistamine. These studies suggest that intravascular activation of the complement system leads to neutrophil aggregation and activation, intrapulmonary capillary sequestration of neutrophils, and vascular injury, which may be related to production of toxic oxygen metabolites by complement-activated neutrophils.

Evidence for role of hydroxyl radical in complement and neutrophil-dependent tissue injury.

Using our recently described model of acute lung injury in rats after systemic activation of complement by cobra venom factor (CVF), we demonstrated that pretreatment of animals with human milk apolactoferrin (in its native or derivatized form), but not iron-saturated lactoferrin, provides significant protection against complement- and neutrophil-mediated lung injury. The synthetic iron chelator deferoxamine mesylate also affords protection from lung injury. The protective effects of apolactoferrin are not related to a blocking of CVF-induced complement activation. We also demonstrated that infusion of ionic iron, especially Fe3+, greatly potentiates lung vascular injury after systemic complement activation. Finally, protection from lung injury occurs in animals pretreated with the potent scavenger of hydroxyl radicals (OH.), dimethyl sulfoxide. Based on transmission electron microscopy, CVF-treated rats show leukoaggregates and endothelial cell destruction in interstitial pulmonary capillaries, along with intraalveolar hemorrhage and fibrin deposition. In animals protected with apolactoferrin, deferoxamine mesylate, or dimethyl sulfoxide, the morphological studies reveal leukoaggregates but no endothelial cell damage, hemorrhage, or fibrin deposition. These data support the concept that tissue injury that is complement and neutrophil dependent may be related to generation of OH. derived from H2O2 after leukocytic activation.

Systemic complement activation, lung injury, and products of lipid peroxidation.

Previously we have demonstrated that systemic activation of the complement system after intravenous injection of cobra venom factor (CVF) results in acute lung injury as reflected by increases in the vascular permeability of the lung as well as by morphologic evidence of damage to lung vascular endothelial cells. In using the vascular permeability of the lung as the reference, the current studies show a quantitative correlation between lung injury and the appearance in plasma of lipid peroxidation products (conjugated dienes) as well as increased concentrations of lactic dehydrogenase (LDH) and one of its isoenzymes (LDH-4). After injection of CVF, extracts of lungs also showed elevated levels of conjugated dienes, whereas no elevations were found in extracts of liver, kidney, and spleen. There was no evidence in CVF-injected rats of renal or hepatic injury as reflected by the lack of development of proteinuria and the failure to detect increased serum levels of liver-related enzymes. Other peroxidation products identified in plasma of CVF-injected rats involved hydroperoxides and fluorescent compounds with features of Schiff bases. Not surprisingly, malondialdehyde was not found to be a reliable plasma indicator of lipid peroxidation associated with oxygen radical-mediated lung vascular injury. In using a model of oxygen radical-independent lung injury induced by oleic acid, although large amounts of LDH and LDH-4 were found in the plasma, no increases in plasma levels of conjugated dienes were detected. In CVF-injected animals treated with interventions protective against lung injury (neutrophil depletion, catalase, hydroxyl radical scavengers, or iron chelators), there were striking reductions in the plasma levels of conjugated dienes, hydroperoxides, and fluorochromic products. Morphometric analysis of lung sections revealed that the protective interventions did not interfere with the accumulation of neutrophils in lung interstitial capillaries after systemic activation of complement. In vitro studies with phorbol-stimulated neutrophils failed to demonstrate appearance of conjugated dienes, suggesting that the dienes appearing in plasma of CVF-injected animals are not the result of autotoxic changes in neutrophils. The data presented in this paper suggest that acute lung injury mediated by oxygen radicals derived from phagocytic cells can be monitored by the appearance in plasma of products of lipid peroxidation.

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.

A major role for neutrophils in experimental bullous pemphigoid.

Bullous pemphigoid (BP) is an inflammatory subepidermal blistering disease associated with an IgG autoimmune response to the hemidesmosomal protein, BP180. Using a passive transfer mouse model, our group has shown previously that antibodies to the murine BP180 (mBP180) ectodomain are capable of triggering a blistering skin disease that closely mimics human BP. In this study, we investigated the role of neutrophils in the immunopathogenesis of this disease model. BALB/c mice depleted of circulating neutrophils by treatment with neutrophil-specific antibodies were no longer susceptible to the pathogenic effects of anti-mBP180 IgG. IgG and complement were deposited at the dermal-epidermal junction of these animals, but there was no evidence of inflammatory infiltration or blistering. C5-deficient mice, which are resistant to the pathogenic activity of anti-mBP180 IgG, could be made susceptible to this IgG-mediated blistering disease by intradermal administration of a neutrophil chemoattractant, IL-8 or C5a. Intraperitoneal injection of IL-8, which sequesters neutrophils in the peritoneal cavity, interferes with anti-mBP180-induced neutrophilic infiltration of the skin and prevented the development of BP disease in BALB/c mice. These findings provide the first direct evidence that neutrophils recruited to the skin via a C5-dependent pathway play an essential role in subepidermal blister formation in experimental BP, and suggest new directions for disease intervention.

Mediator-induced activation of xanthine oxidase in endothelial cells.

Rat pulmonary artery endothelial cells incubated with human serum that has been complement‐activated by addition of cobra venom factor reveal a pronounced conversion of xanthine dehydrogenase to xanthine oxidase. This process requires the availability of the fifth component of complement (C5) but not the presence of other components (C2 and C6‐C9). The phenomenon can be reproduced by addition to endothelial cells of purified human recombinant C5a but not C5a desArg or C3a. The enzyme conversion process is relatively rapid (occurring within 5‐10 min), requires the presence of intact endothelial cells, and does not require protein synthesis. Similar effects on endothelial cells have been obtained with human recombinant tumor necrosis factor a and the chemotactic peptide N‐formyl‐Met‐Leu‐Phe. In contrast, bradykinin, recombinant human interleukin 1β, and phorbol ester lack this biological activity. These findings suggest novel effects of inflammatory mediators on endothelial cells.— Friedl, H. P.; Till, G. O.; Ryan, U. S.; Ward, P. A. Mediator‐induced activation of xanthine oxidase in endothelial cells. FASEB J. 3: 2512‐2518; 1989.

Oxygen Radical Dependent Lung Damage following Thermal Injury of Rat Skin

Acute thermal injury (70 degrees C, 30 sec) to rat skin results in progressive consumptive depletion of the complement system. Individual complement components (C3, C4, C6) each show reductions in hemolytic activity. Crossed immunoelectrophoresis analysis of serum from thermally injured rats reveals conversion of C3 compatible with activation of the complement system. During the first hour following thermal injury, C5a-related chemotactic activity appears in the serum and is temporally related to the development of neutropenia. Lung injury, as revealed by increases in lung permeability, develops progressively during a 6-hour period and parallels changes in complement levels. Morphologically, lung changes include leukoaggregates within pulmonary capillaries and the presence of intra-alveolar hemorrhage. Protection from lung injury following remote thermal injury to skin is afforded by depleting animals of complement or neutrophils, or by systemic treatment of animals with a combination of catalase and superoxide dismutase. Antihistamine drugs have no protective effect. These data suggest that acute thermal injury leads to systemic complement activation, neutrophil activation, and acute lung injury that is related to production of toxic oxygen products by activated blood neutrophils.

Correlation of the local and systemic cytokine response with clinical outcome following thermal injury.

Eighty-eight patients with acute thermal injury were evaluated. Forty-eight hours after injury, TNF, IL-6, and IL-8 were significantly present in the systemic circulation, lung, normal skin, and thermally injured skin. The presence of TNF, IL-6, and IL-8 proteins in the lung, normal skin, and thermally injured skin were associated with TNF, IL-6, and IL-8 mRNA upregulation. Logistic regression analysis controlling for the Abbreviated Burn Severity Index demonstrated that the presence of IL-8 in the lung was associated with early pulmonary physiologic dysfunction (p = 0.006) and nosocomial pulmonary infection (p = 0.040). We conclude that acute thermal injury initiates an early systemic, lung, and skin response involving TNF, IL-6, and IL-8. The TNF, IL-6, and IL-8 protein present in the lung and skin in response to acute thermal injury are generated locally and do not originate from the systemic cytokine pool. The lung cytokine response to acute thermal injury may initiate local organ failure.

Pathophysiologic Events Related to Thermal Injury of Skin

Acute thermal injury of skin equivalent to second-degree injury and involving approximately 25% of total body surface results in a series of pathophysiologic events which lead to both local and distant tissue/organ injury. The distant effects involve intravascular hemolysis and acute lung injury, both of which can be attributed to complement activation and intravascular stimulation of neutrophils, resulting in oxygen radical production, which results in injury of red cells and pulmonary vascular endothelial cells. At the local site of thermal injury, the progressive increase in vascular permeability is linked to complement activation and histamine release, the outcome of which is interaction of histamine with xanthine oxidase, resulting in enhanced catalytic activity of the enzyme. Toxic oxygen products of xanthine oxidase, including H2O2 and its conversion product, the hydroxyl radical, appear to be linked to the damage of dermal vascular endothelial cells, resulting in progressive vascular permeability. The increased vascular permeability can be greatly attenuated by the use of inhibitors of xanthine oxidase, the inhibitor of histamine release (cromolyn), catalase, an iron chelator (deferoxamine), or scavengers of the hydroxyl radical. Interestingly, neutrophils appear to play little if any role in dermal vascular injury in this animal model of thermal trauma. Those studies suggest that pathophysiologic events following local thermal trauma are complex and involve a variety of mediator pathways.

The complement system

1 Components and Reactivity.- 1.1 Components.- 1.1.1 Factors of the Classical Pathway.- 1.1.2 Components of the Alternative Pathway.- 1.1.3 Late Components.- 1.2 Reactivity.- 1.2.1 Classical Pathway of Activation.- 1.2.2 Lectin Pathway of Non-self Recognition.- 1.2.3 Alternative Pathway: Activation and Regulation.- 1.2.4 Complement Attack Phase.- 1.2.5 Control Mechanisms.- 1.2.5.1 Membrane Cofactor Protein (CD46) and Decay-Accelerating Factor (CD55).- 1.2.5.2.1 Control of C5b-9 by Fluid Phase Factors.- 1.2.5.2.2 Membrane-Bound Inhibitors of C5b-9.- 1.2.5.3 Interspecies Incompatibilities of Complement Factors and of Regulators.- 1.3 Surface Receptors and Signaling Pathways.- 1.3.1 Receptor for C1q.- 1.3.2 Receptors for Human C3 Fragments.- 1.3.3 Receptors for C5a, C3a, and Factor H.- 2 Biologic Functions.- 2.1 Complement in the Induction of Antibody Response.- 2.2 Maintenance of Immune Complex Solubility and Immune Adherence.- 2.3 Interaction with Effector Cells.- 2.3.1 Leukocyte Mobilisation/Recruitment.- 2.3.2 Chemotactic Peptides.- 2.3.3 Cellular Responses to Activation Products.- 2.4 Host Defense Against Infection.- 2.4.1 Defense Against Bacteria.- 2.4.2 Complement-Dependent Virus Neutralization.- 2.4.3 Evasion of Complement-Mediated Damage by Microorganisms.- 2.5 Possible Role of Complement Regulators in Reproduction.- 2.6 Network Interactions of the Complement System with Other Serum Mediator Systems.- 3 Pathology.- 3.1 Complement Deficiencies in Animals: Impact on Biological Functions.- 3.2 Complement Deficiencies in Humans.- 3.2.1 Inherited and Acquired Deficiencies of C1 Esterase Inhibitor in Humans.- 3.2.2 Deficiencies in the Classiral Pathway.- 3.2.3 Deficiencies in the Alternative Pathway: Factors I and H.- 3.2.4 Deficiency in Terminal Reactivity.- 3.2.5 Deficiency in Lysis Control Proteins.- 3.2.6 C3 Receptor Deficiencies.- 3.3 C3 Nephritic Factor.- 3.4 Complement in Inflammation.- 3.5 Role of Complement in Graft Rejection.- 3.6 Complement Activation on Artificial Surfaces in Biomedical Therapies.- 3.7 Adverse Reactions to Drugs.- 4 Complement Manipulation In Vivo.- 5 The Clinical Laboratory: Testing the Complement System.