Boris J. Czermak

Inhibition of NF-κB activation and augmentation of IκBβ by secretory leukocyte protease inhibitor during lung inflammation

In earlier experiments, exogenous administration of secretory leukocyte protease inhibitor (SLPI) suppressed acute lung injury induced by deposition of IgG immune complexes. In the current studies we examined the mechanism of the protective effects of SLPI in this model. The presence of SLPI in the IgG immune complex-model of lung injury reduced the increase in extravascular leakage of 125I-albumin, the intensity of up-regulation of lung vascular intercellular adhesion molecule-1, and the numbers of neutrophils accumulating in the lung. The presence of SLPI caused greatly reduced activation (ie, nuclear translocation) of the transcription nuclear factor-κB (NF-κB) in lung cells but did not suppress activation of lung mitogen-activated protein kinase. SLPI did not alter NF-κB activation in alveolar macrophages harvested 30 minutes after initiation of lung inflammation. In the presence of SLPI, content of tumor necrosis factor-α, CXC chemokines, and C5a in bronchoalveolar fluids was unaffected. In the inflamed lungs, inhibition of NF-κB activation by SLPI was associated with elevated levels of lung IκBβ (but not IκBα) protein in the absence of elevated mRNA for IκBβ. When instilled into normal lung, SLPI also caused similar changes (increases) in lung IκBβ. Finally, in the lung inflammatory model used, the presence of anti-SLPI caused accentuated activation of NF-κB. These data confirm the anti-inflammatory effect of SLPI in lung and point to a mechanism of anti-inflammatory effects of SLPI. SLPI appears to function as an endogenous regulator of lung inflammation.

Role of CC Chemokines (Macrophage Inflammatory Protein-1β, Monocyte Chemoattractant Protein-1, RANTES) in Acute Lung Injury in Rats

The role of the CC chemokines, macrophage inflammatory protein-1β (MIP-1β), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1β, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1β and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1β Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-α in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1β, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1β, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1β, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.

Mechanisms of Enhanced Lung Injury during Sepsis

A major complication in sepsis is progressively impaired lung function and susceptibility to intrapulmonary infection. Why sepsis predisposes the lung to injury is not clear. In the current studies, rats were rendered septic by cecal ligation/puncture and evaluated for increased susceptibility to injury after a direct pulmonary insult (deposition of IgG immune complexes or airway instillation of lipopolysaccharide). By itself, cecal ligation/puncture did not produce evidence of lung injury. However, after a direct pulmonary insult, lung injury in septic animals was significantly enhanced. Enhanced lung injury was associated with increased accumulation of neutrophils in lung, enhanced production of CXC chemokines (but not tumor necrosis factor-alpha) in bronchoalveolar lavage fluids, and increased expression of lung vascular intercellular adhesion molecule-1 (ICAM-1). Complement depletion or treatment with anti-C5a abolished all evidence of enhanced lung injury in septic animals. When stimulated in vitro, bronchoalveolar lavage macrophages from septic animals had greatly enhanced CXC chemokine responses as compared with macrophages from sham-operated animals or from septic animals that had been complement depleted. These data indicate that the septic state causes priming of lung macrophages and suggest that enhanced lung injury in the septic state is complement dependent and related to increased production of CXC chemokines.

Roles for C-X-C chemokines and C5a in lung injury after hindlimb ischemia-reperfusion

We evaluated the roles of the C-X-C chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) as well as the complement activation product C5a in development of lung injury after hindlimb ischemia-reperfusion in rats. During reperfusion, CD11b and CD18, but not CD11a, were upregulated on neutrophils [bronchoalveolar lavage (BAL) and blood] and lung macrophages. BAL levels of CINC and MIP-2 were increased during the ischemic and reperfusion periods. Treatment with either anti-CINC or anti-MIP-2 IgG significantly reduced lung vascular permeability and decreased lung myeloperoxidase content by 93 and 68%, respectively (P < 0.05). During the same period, there were significant increases in serum C5a-related neutrophil chemotactic activity. Treatment with anti-C5a decreased lung vascular permeability, lung myeloperoxidase, and BAL CINC by 51, 58, and 23%, respectively (P < 0.05). The data suggest that the C-X-C chemokines CINC and MIP-2 as well as the complement activation product C5a are required for lung neutrophil recruitment and full induction of lung injury after hindlimb ischemia-reperfusion in rats.We evaluated the roles of the C-X-C chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) as well as the complement activation product C5a in development of lung injury after hindlimb ischemia-reperfusion in rats. During reperfusion, CD11b and CD18, but not CD11a, were upregulated on neutrophils [bronchoalveolar lavage (BAL) and blood] and lung macrophages. BAL levels of CINC and MIP-2 were increased during the ischemic and reperfusion periods. Treatment with either anti-CINC or anti-MIP-2 IgG significantly reduced lung vascular permeability and decreased lung myeloperoxidase content by 93 and 68%, respectively ( P < 0.05). During the same period, there were significant increases in serum C5a-related neutrophil chemotactic activity. Treatment with anti-C5a decreased lung vascular permeability, lung myeloperoxidase, and BAL CINC by 51, 58, and 23%, respectively ( P < 0.05). The data suggest that the C-X-C chemokines CINC and MIP-2 as well as the complement activation product C5a are required for lung neutrophil recruitment and full induction of lung injury after hindlimb ischemia-reperfusion in rats.

Role of complement in in vitro and in vivo lung inflammatory reactions

Complement is one of the integral buttresses of the inflammatory response. In addition to host defense activities, proinflammatory properties of several complement components are described. This overview elucidates the role of complement in inflammatory reactions in vitro and in vivo, focusing on the complement activation products, C5a, and the membrane attack complex, C5b‐9. Using several approaches, the impact of these complement components in mechanisms relevant to neutrophil recruitment is emphasized. In addition, the participation of complement in endothelial superoxide generation and its essential requirement for full expression of lung injury is demonstrated, as are the involved intracellular signal transduction pathways. Understanding the mechanisms of complement‐induced proinflammatory effects may provide a basis for future therapeutic blockade of complement and/or its activation products. J. Leukoc. Biol. 64: 40–48; 1998.

Synergistic enhancement of chemokine generation and lung injury by C5a or the membrane attack complex of complement.

Complement plays an important role in many acute inflammatory responses. In the current studies it was demonstrated that, in the presence of either C5a or sublytic forms of the complement-derived membrane attack complex (MAC), rat alveolar macrophages costimulated with IgG immune complexes demonstrated synergistic production of C-X-C (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) and C-C (macrophage inflammatory protein-1alpha and monocyte chemoattractant-1) chemokines. In the absence of the costimulus, C5a or MAC did not induce chemokine generation. In in vivo studies, C5a and MAC alone caused limited or no intrapulmonary generation of chemokines, but in the presence of a costimulus (IgG immune complexes) C5a and MAC caused synergistic intrapulmonary generation of C-X-C and C-C chemokines but not of tumor necrosis factor alpha. Under these conditions increased neutrophil accumulation occurred, as did lung injury. These observations suggest that C5a and MAC function synergistically with a costimulus to enhance chemokine generation and the intensity of the lung inflammatory response.

Differing Patterns of P-Selectin Expression in Lung Injury

Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression was sustained for the next 7 hours, in striking contrast to the pattern of P-selectin expression in the cobra venom factor model, in which upregulation was very transient (within the 1st hour). In the immune complex model, injury and neutrophil accumulation were P-selectin dependent. Upregulation of P-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor alpha. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl sulfoxide sensitive but neutrophil independent. Different mechanisms that may explain why upregulation of lung vascular P-selectin is either transient or sustained are discussed.

REGULATORY EFFECTS OF INTERLEUKIN-11 DURING ACUTE LUNG INFLAMMATORY INJURY

The role of interleukin‐11 (IL‐11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL‐11 mRNA and protein were both up‐regulated during the course of this inflammatory response. Exogenously administered IL‐11 substantially reduced, in a dose‐dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti‐inflammatory effects of IL‐11 were associated with reduced NF‐κB activation in lung, reduced levels of tumor necrosis factor α (TNF‐α) in bronchoalveolar lavage (BAL) fluids, and diminished up‐regulation of lung vascular ICAM‐1. It is interesting that IL‐11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein‐2 (MIP‐2) and cytokine‐inducible neutrophil chemoattractant (CINC); the presence of IL‐11 did not affect these chemokines. However, BAL content of C5a was reduced by IL‐11. These data indicate that IL‐11 is a regulatory cytokine in the lung and that, like other members of this family, its anti‐inflammatory properties appear to be linked to its suppression of NF‐κB activation, diminished production of TNF‐α, and reduced up‐regulation of lung vascular ICAM‐1. J. Leukoc. Biol. 66: 151–157; 1999.

Intramedullary nailing technique in polytrauma

Da Frakturen langer Röhrenknochen eine wesentliche additive Systembelastung durch Schmerzen, Blutverlust und Mediatorenfreisetzung bewirken und instabile Verhältnisse im Bereich der Extremitäten die ordnungsgemäße Intensivbehandlung polytraumatisierter Patienten nachhaltig behindern, besteht heute über die Notwendigkeit der Primärstabilisierung langer Röhrenknochen bei schwer verletzten Patienten weitgehend Einigkeit. Doch auch nach jahrzehntelanger Diskussion und Forschung gehen die Meinungen über das optimale Fixationsverfahren in der Akutphase weit auseinander. Bei der Indikationsstellung zur primären Marknagelung bei Polytraumatisierten sind neben Erkenntnissen aus wissenschaftlichen Untersuchungen auch logistische Erwägungen zu berücksichtigen: Gerade die ungebohrten Techniken sind anspruchsvoll, weswegen unter ungünstigen Bedingungen bezüglich Personal, Tageszeit und Equipment durchaus suboptimale Ergebnisse unter erheblich erhöhtem Zeitaufwand resultieren können. Daher sind bei bestimmten Verletzungsmustern und ab einer gewissen Verletzungsschwere auch aus logistischen Gründen Vor- und Nachteil der primären Stabilisierung durch Marknageltechniken abzuwägen. Hier kann ggf. die primäre Stabilisierung mittels Fixateur externe in Folge der unkomplizierten und raschen Anwendung dem Patienten deutliche Vorteile bringen: Ein geringeres additives operatives Trauma in der initialen vulnerablen Phase sowie kürzere Operationszeiten und ein geringerer Bedarf an Blutersatzstoffen können synergistisch mit der früher einsetzenden intensivmedizinischen Weiterversorgung entscheidende Vorteile bringen. In der sekundären Operationsphase (nach dem 4. posttraumatischen Tag) kann dann in der Regel einzeitig auf die biomechanisch überlegene Marknagelung umgestiegen werden. Wird die primäre Marknagelung angestrebt, sind die Aspekte einer zusätzlichen Belastung zu berücksichtigen, wie z. B. extreme Markraumbohrungen, die Verwendung stumpfer Bohrer oder die hohe Druckentwicklung beim Eintreiben solider Marknägel in dünnlumige Markhöhlen bei einfachen Frakturtypen. Der ansonsten gesunde Mensch mit einer unverletzten Lunge kann die resultierenden Fett- und Partikelembolisationen noch kompensieren. Für den entsprechend vorbelasteten Polytraumatisierten, dessen Defensivsysteme bereits bis an die Kompensationsgrenze belastet sind, können diese iatrogenen Belastungen jedoch zu irreversiblen Schädigungen bis hin zu einem letalen Ausgang führen.As fractures of the long bones cause a substantial additive systemic stress by way of pain, blood loss and release of mediators, and instability of the extremities means that the intensive treatment that polytraumatized patients need can be delayed for a long time, the necessity for primary stabilization of long bones in severely injured patients is now generally accepted. After decades of discussion and research, however, there are still wide differences of opinion on which fixation technique is best in the acute phase. When polytraumatized patients are considered to decide whether primary medullary nailing is indicated logistic aspects should be taken into account as well as information derived from scientific investigations: The techniques making use of unreamed nails are challenging to the surgeon, which means that if conditions are not completely favourable in terms of staff, time of day and equipment it is certainly possible for a suboptimal outcome to be obtained with a substantially greater investment of time than normal. Thus, there are also logistic reasons why, in the presence of certain injury patterns and from a certain level of severity of the injuries onward, the advantages and disadvantages of primary stabilization by medullary nailing techniques should be carefully weighed against each other. In these circumstances primary stabilization with an external fixator can have obvious advantages for the patient, as it is so uncomplicated and quick to apply. A lesser additive trauma from the operation in the initial phase when the patient is so vulnerable, plus the shorter operating times and the lower requirement for blood substitutes can have decisive advantages in synergy with the earlier start of medical intensive care. In the secondary surgical phase (more than 4 days after the trauma) it is then generally possible to convert to intramedullary nailing, which gives a superior biomechanical result, in a single operative session. If primary intramedullary nailing is attempted, the possibility of additional stress should be borne in mind; this can be caused, for example, by the use of blunt drills or the high pressure that can develop when solid nails are driven into narrow lumina in the case of simple fractures. An otherwise healthy person with undamaged lungs can compensate for the ¶resulting fat and particle emboli. In the ¶case of a multiply injured patient who is already in a badly weakened state with defence systems strained to the limit, however, such iatrogenic stresses can lead to ¶irreversible damage and even to a lethal ¶outcome.

Use of Animal Models to Define Complement Functions

There appears to be little doubt that the complement system and its activation products play important roles in expression of both immune and inflammatory responses. Defining of the role of the complement system, especially in the context of the inflammatory response, has been deduced on the basis of strategies listed in Table 1. The use of laboratory animals with naturally occurring genetic deficiencies of complement components has provided important clues into the biological roles of complement. Guinea pigs with a genetic deficiency of C4 have defective clearance of intravenously infused sheep red blood cells containing IgG antibody directed toward surface epitopes (1,2). This defect can be corrected by restoration of whole hemolytic activity of serum complement (CH50), at which time clearance of sensitized red cells becomes very rapid, presumably through engagement of complement receptors on phagocytic cells of the spleen and liver. Rabbits with genetic deficiency of C6 exhibit acute inflammatory vasculitis in the reversed passive Arthus reaction in skin, the intensity of the reaction being similar to the inflammatory response C6-intact rabbits, but with less hemorrhage in thr skin lesions in the absence of C6 (3).