Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats.

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), 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-1 beta, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta 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-1 beta 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-alpha 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-1 beta, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1 beta, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1 beta, 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.

Association between endometrial cancer and tamoxifen treatment of breast cancer.

A retrospective cohort-study in 4109 breast cancer patients was undertaken to determine how tamoxifen affected the risk of endometrial cancer. Data on 1701 tamoxifen-treated women were analysed. Two thousand four hundred and eight non-tamoxifen users served as control group. The occurrence of new primary uterine cancers was assessed by computerized linkage to the Austrian Cancer Registry. Twenty-five women who subsequently developed endometrial cancer were identified. Eight uterine cancers occurred in the tamoxifen group, whereas 17 uterine cancers were found in the control group. The estimate of the relative risk (RR) showed an increased risk to develop endometrial cancer for the tamoxifen group RR 1.136 (95% CI 0.71; 1.80). Analysis of relevant confounding variables did not show any differences in the two groups. In conclusion, this retrospective study demonstrated a non-significant increased risk of endometrial cancer in women receiving tamoxifen as treatment for breast cancer. However, the magnitude of RR and the absolute number of endometrial cancer cases in this long term observation demonstrate clearly that the clinical benefit of tamoxifen therapy greatly outweighs the risk.

Protective effects of C5a blockade in sepsis.

Sepsis in humans is a difficult condition to treat and is often associated with a high mortality rate. In this study, we induced sepsis in rats using cecal ligation and puncture (CLP). In rats depleted of the complement factor C3, CLP led to very short survival times (about 4 days). Of the rats that underwent CLP ('CLP rats') that were C3-intact and treated with preimmune IgG, most (92%) were dead by 7 days. Blood neutrophils from these rats contained on their surfaces the powerful complement activation product C5a. This group had high levels of bacteremia, and their blood neutrophils when stimulated in vitro had greatly reduced production of H2O2, which is known to be essential for the bactericidal function of neutrophils. In contrast, when companion CLP rats were treated with IgG antibody against C5a, survival rates were significantly improved, levels of bacteremia were considerably reduced, and the H2O2 response of blood neutrophils was preserved. Bacterial colony-forming units in spleen and liver were very high in CLP rats treated with preimmune IgG and very low in CLP rats treated with IgG antibody against C5a, similar to values obtained in rats that underwent 'sham' operations (without CLP). These data indicate that sepsis causes an excessive production of C5a, which compromises the bactericidal function of neutrophils. Thus, C5a may be a useful target for the treatment of sepsis.

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.

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.

In vitro and in vivo dependency of chemokine generation on C5a and TNF-alpha.

Under a variety of conditions, alveolar macrophages can generate early response cytokines (TNF-alpha, IL-1), complement components, and chemotactic cytokines (chemokines). In the current studies, we determined the requirements for TNF-alpha and the complement activation product C5a in chemokine production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro was studied in rat alveolar macrophages stimulated with IgG immune complexes in the absence or presence of Abs to TNF-alpha or C5a. The rat lung injury model induced by IgG immune complex deposition was employed for in vivo studies. Abs to TNF-alpha or C5a were administered intratracheally or i.v., and effects on chemokine levels in bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro and in vivo studies demonstrated the requirements for TNF-alpha and C5a for full generation of CXC and CC chemokines. In vitro and in vivo blockade of TNF-alpha or C5a resulted in significantly reduced production of chemokines. Supernatant fluids from in vitro-stimulated macrophages revealed by Western blot analysis the presence of C5a/C5adesArg, indicating intrinsic generation of C5a/C5adesArg by alveolar macrophages and explaining the higher efficiency of intratracheal vs i.v. blockade of C5a in reducing chemokine production. These results underscore the central role of both TNF-alpha and C5a, which appear to function as autocrine activators to promote CXC and CC chemokine generation by alveolar macrophages.

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.

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.

Soluble ICAM-1 activates lung macrophages and enhances lung injury.

Because of the important role of rat ICAM-1 in the development of lung inflammatory injury, soluble recombinant rat ICAM-1 (sICAM-1) was expressed in bacteria, and its biologic activities were evaluated. Purified sICAM-1 did bind to rat alveolar macrophages in a dose-dependent manner and induced production of TNF-alpha and the CXC chemokine, macrophage inflammatory protein-2 (MIP-2). Alveolar macrophages exhibited cytokine responses to both sICAM-1 and immobilized sICAM-1, while rat PBMCs failed to demonstrate similar responses. Exposure of alveolar macrophages to sICAM-1 resulted in NFkappaB activation (which was blocked by the presence of the aldehyde peptide inhibitor of 28S proteosome and by genistein, a tyrosine kinase inhibitor). As expected, cross-linking of CD18 on macrophages with Ab resulted in generation of TNF-alpha and MIP-2. This response was also inhibited in the presence of the proteosome inhibitor and by genistein. Alveolar macrophages showed adherence to immobilized sICAM-1 in a CD18-dependent manner. Finally, airway instillation of sICAM-1 intensified lung injury produced by intrapulmonary deposition of IgG immune complexes in a manner associated with enhanced lung production of TNF-alpha and MIP-2 and increased neutrophil recruitment. Therefore, through engagement of beta2 integrins, sICAM-1 enhances alveolar macrophage production of MIP-2 and TNF-alpha, the result of which is intensified lung injury after intrapulmonary disposition of immune complexes.

Complement, cytokines, and adhesion molecule expression in inflammatory reactions.

Directional emigration of neutrophils is the hallmark of phlogistic processes. The involved regulatory mechanisms include complement activation products, cytokines, adhesion molecules, and their respective counter receptors. In this article, we discuss the role of these mediators in initiation, development, and containment of acute inflammatory responses in in vitro and in vivo models, showing interactions between a network of proinflammatory and anti-inflammatory effectors. A major focus deals with the effects of complement on activation of the vascular endothelium. In addition, the role of interleukins in the regulation of inflammatory responses is described.

Characterization of rat lung ICAM-1.

OBJECTIVE AND DESIGN: We expressed soluble rat ICAM-1, generated a polyclonal anti-ICAM-1 antibody, and studied ICAM-1 upregulation in lung inflammatory conditions. Bacterial and baculovirus expression systems were employed. MATERIAL: 250 g adult, male Long Evans rats were used. For in vitro studies, rat pulmonary artery endothelial cells (RPAEC), rat alveolar macrophages and aortic rings were stimulated (as described below) and evaluated for ICAM-1 expression. TREATMENT: RPAEC and macrophages were stimulated with lipopolysaccharide (LPS) and recombinant murine tumour necrosis factor alpha (TNFalpha). In vivo immunoglobulin G (IgG) immune complex-induced lung injury was employed. METHODS: Enzyme-linked immunoassay (ELISA) Western and Northern blot analyses and immunohistochemical evaluations were performed. All experiments were done at least in duplicate. Data were analyzed by two-tailed Student's t-test. RESULTS: ICAM-1 expression of RPAEC was time- and dose-dependent, peaking at 6h after LPS-stimulation. LPS and TNFalpha each enhanced ICAM-1 expression on alveolar macrophages (reaching a maximum at 2 h). In IgG immune complex-induced lung injury, ICAM-1 mRNA isolated from whole lung peaked at 4 h, while lung ICAM- I protein peaked at 6 h. CONCLUSIONS: Quantitation of ICAM-1 expression in vitro and in vivo suggests that ICAM-1 plays a central role in two lung inflammatory models. Furthermore, lung ICAM-1 upregulation involves at least two cell types: vascular endothelial cells and alveolar macrophages.

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.

Streptococcal cell wall-induced arthritis: requirements for IL-4, IL-10, IFN-gamma, and monocyte chemoattractant protein-1.

Intra-articular injection of streptococcal cell wall Ag followed by i.v. challenge ("reactivation") results in a destructive lymphocyte-dependent monoarticular arthritis. To further define the role of immune mechanisms in the model, Abs to Th1 and Th2-related cytokines were evaluated. Treatment of rats with antibodies to IL-4 reduced swelling, while treatment with anti-IL-10 or anti-IFN-gamma either had no effect or slightly enhanced the inflammatory response. These results suggest that Th-2 immune mechanisms may be, at least in part, operative in the model. To more precisely define the role of IL-4, the effects of anti-IL-4 on monocyte chemoattractant protein-1 (MCP-1) expression were evaluated. Initial studies demonstrated that mRNA (as determined by in situ hybridization) and protein (as determined by immunofluorescence) for MCP-1 were detectable in inflamed synovial tissue in a time-dependent manner. Anti-IL-4 treatment significantly reduced the expression of mRNA for MCP-1 24 and 72 h after reactivation. In addition, anti-MCP-1 inhibited swelling and reduced influx of (111)In-labeled T cells. These data suggest that the reactivation model of streptococcal cell wall Ag-induced arthritis is Th-2 dependent, and that an inter-relationship exists between IL-4 and the expression of MCP-1.

[Fracture management during bone growth]. / Frakturversorgung am wachsenden Skelett.

In treating fractures in children, there have been more and more demands for the primary treatment to be definitively planned, i.e., for a conclusive order of events to be laid down so as to exclude the necessity for re-reductions or a change of therapy. Long-term conservative methods of treatment are becoming less and acceptable to parents and children. From the psychosocial point of view, the aim must be to avoid extensive periods of hospitalization or bedrest in traction and to dispense with repeated anesthesia for re-reduction and changes in procedure. The child should be able to leave the hospital as soon as possible after a definitive and circumscribed primary course of treatment. Since complication-free surgical interventions are now available, many more operative procedures for dealing with fractures during the years of growth have become routine in this field.

Expression of lung vascular and airway ICAM-1 after exposure to bacterial lipopolysaccharide.

Airway instillation of bacterial lipopolysaccharide (LPS) into rat lungs induces neutrophil accumulation, which is known to be intercellular adhesion molecule-1 (ICAM-1)-dependent. In the present study, ICAM-1 messenger RNA (mRNA) of whole lung was found to increase by 20-fold in this inflammatory model. This increase was reduced by 81% after treatment of animals with anti-tumor necrosis factor-alpha (TNF-alpha) antibody and by 37% after treatment with anti-interleukin-1 (IL-1) antibody. The same interventions reduced whole-lung ICAM-1 protein by 85% and 25%, respectively. The studies were extended to assess the locale in lung of ICAM-I upregulation. Lung vascular ICAM-1 content, which was assessed by vascular fixation of [125I]anti-ICAM-1, rose 4-fold after airway instillation of LPS. This rise was also TNF-alpha-dependent. Under the same experimental conditions, fixation of [125I]anti-ICAM-1 to airway surfaces increased 11-fold in a TNF-alpha-dependent manner. In situ hybridization and immunohistochemical analyses of lung tissue revealed ICAM-1 upregulation in the bronchiolar epithelium and in peribronchiolar smooth muscle. Soluble ICAM-1 could also be detected in bronchoalveolar lavage fluids (BALFs) of animals after intratracheal instillation of LPS. Retrieved alveolar macrophages showed a small, significant, and transient increase in surface expression of ICAM-1. These data indicate, at the very least, a dual compartmentalized (vascular and airway) upregulation of ICAM-1 after airway instillation of LPS. This upregulation requires TNF-alpha and IL-1. The functional significance of upregulated airway ICAM-1 remains to be determined.

Streptococcal cell wall-induced arthritis. Requirements for neutrophils, P-selectin, intercellular adhesion molecule-1, and macrophage-inflammatory protein-2.

Immune arthritis in rat ankle joints was induced by intra-articular injection of streptococcal cell was extract (SCW), followed 21 days later by i.v. injection of SCW. This results in a monoarticular arthritis characterized by an influx of neutrophils and mononuclear cells, a 35-fold increase in urinary excretion of 8-hydroxy-deoxyguanosine (8-OH-dGUA; an index of free radical production), ankle edema, and joint damage/destruction. Neutrophil depletion substantially reduced the intensity of ankle edema. Ab-induced blockade of P-selectin or ICAM-1 also reduced the intensity of ankle edema and the influx of neutrophils. Blockade of TNF-alpha or IL-1 resulted in nearly complete and persistent reduction in ankle edema and profound reductions in the accumulation of neutrophils and mononuclear cells in affected joints. Finally, blocking of macrophage-inflammatory protein-2 reduced ankle edema and neutrophil accumulation during the first 2 days after i.v. challenge with SCW. These data indicate that SCW-induced arthritis is neutrophil dependent and that the recruitment of neutrophils and subsequent joint edema requires ICAM-1, P-selectin, and macrophage-inflammatory protein-2, as well as TNF-alpha and IL-1.

Inflammatory response after abdominal trauma, infection, or intestinal obstruction measured by oxygen radical production in peritoneal fluid.

BACKGROUND: Reactive oxygen intermediates (ROI) have been implicated in many pathophysiological processes of inflammatory tissue damage and tissue repair. In the present study we compared the production of ROI in three different types of tissue damage in surgical patients. METHODS: Peritoneal fluid specimens were harvested during the initial operation and postoperatively from 25 surgical patients with abdominal trauma, intraabdominal infection, and intestinal obstruction. The optical density at 412 nm, representing the peroxidation of hemoglobin, was measured to assess intraperitoneal ROI production. Patients were categorized into 3 groups: (A) infected patients with good outcome, (B) patients after trauma or obstruction with good outcome, and (C) patients with poor outcome due to persistent or secondary infection and multiple organ failure. Analysis of variance (ANOVA) and paired t test were used for statistical analysis. RESULTS: Overall, the ROI production decreased significantly at days 2 and 3 compared with day 0 and 1 (P = 0.0013). No initial differences of intraoperative ROI concentrations were found among the three groups; however, patients with a poor outcome showed increased ROI values after 4 to 5 days (P = 0.038) when compared with the good outcome group. CONCLUSIONS: We have demonstrated that intraperitoneal ROI production (1) can be measured in patients with intraabdominal tissue damage, (2) is not different between patients with intraabdominal infections, abdominal trauma, or intestinal obstruction, and (3) correlates with the clinical picture and the presence of an inflammatory intraabdominal focus or tissue damage.

Requirement for C5a in lung vascular injury following thermal trauma to rat skin.

Previous studies in rats have shown that deep second degree dermal burns, involving 28-30% of total body surface area, result in systemic complement activation, appearance in plasma of chemotactic activity, sequestration of blood neutrophils in lung capillaries, and development of neutrophil-dependent dermal vascular and lung vascular injury. Although blockade of complement activation or depletion of complement before skin burns has resulted in significant attenuation of tissue injury both locally and distally (in lung), a role for C5a in these events is unclear. In the following studies, we demonstrate the presence of C5a and neutrophil chemotactic activity in serum and in lung homogenates after thermal injury. C5a has also been found in bronchoalveolar lavage fluids of thermally injured animals. Treatment of animals with a polyclonal neutralizing rabbit antibody to rat C5a was lung protective. The protective effects of the antibody (anti-C5a) were associated with diminished vascular permeability changes, as well as reduced tissue build-up of myeloperoxidase. Anti-C5a also prevented up-regulation of lung vascular ICAM-1 (intercellular adhesion molecule-1) in skin-burned rats. These observations indicate that C5a is essential for development of neutrophil accumulation and vascular permeability increases in distant (lung) organs after thermal trauma to skin. The protective effects of anti-C5a in lung, appear to be related to prevention of up-regulation of vascular ICAM-1. Accordingly, C5a may represent a target for clinical approaches in the treatment of organ injury following thermal trauma.

Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation.

Activation of the complement cascade and subsequent assembly of the membrane attack complex (MAC) occur in a number of pathophysiological settings. When formed on the surface of endothelial cells in sublytic concentrations, the MAC can induce a number of proinflammatory activities, including the secretion of soluble mediators (eg, interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) and the up-regulation of cell surface adhesion molecules. Available data indicate that MAC-induced cell activation may occur through several complex signal transduction pathways, but little is known about the intranuclear mechanisms by which complement-derived products promote the up-regulation of inflammatory mediators. Using purified distal complement proteins (C5-9) to assemble functional MAC on early-passage human umbilical vein endothelial cells (HUVECs), we examined mechanisms of MCP-1 and IL-8 induction. Formation of sublytic concentrations of MAC promoted an increase in nuclear factor (NF)-kappa B DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay. Cytosolic to nuclear translocation of NF-kappa B was confirmed by Western immunoblot and immunocytochemical analyses. Formation of the C5b-8 complex also promoted NF-kappa B translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear translocation of the p65 NF-kappa B subunit was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. Preincubation of HUVECs with pyrrolidine dithiocarbamate prevented MAC-induced increases in IL-8 and MCP-1 mRNA concentrations and protein secretion. A direct cause and effect linkage between MAC assembly and NF-kappa B activation was established through examination of the pharmacological effect of the peptide SN50 on IL-8 and MCP-1 expression. SN50 is a recently engineered 26-amino-acid peptide that contains a lipophilic cell-membrane-permeable motif and a nuclear localization sequence that specifically competes with the nuclear localization sequence of the NF-kappa B p50 subunit. This study provides direct in vitro evidence that the distal complement system (MAC) can promote proinflammatory endothelial cell activation, specifically, increases in IL-8 and MCP-1 mRNA concentrations and protein secretion, and that cytosolic to nuclear translocation of NF-kappa B is necessary for this response.