The complement system was discovered a century ago as a potent defense cascade of innate immunity. After its first description, continuous experimental and clinical research was performed, and three canonical pathways of activation were established. Upon activation by traumatic or surgical tissue damage, complement reveals beneficial functions of pathogen and danger defense by sensing and clearing injured cells. However, the latest research efforts have provided a more distinct insight into the complement system and its clinical subsequences. Complement has been shown to play a significant role in the pathogenesis of various inflammatory processes such as sepsis, multiorgan dysfunction, ischemia/reperfusion, cardiovascular diseases and many others. The three well-known activation pathways of the complement system have been challenged by newer findings that demonstrate direct production of central complement effectors (for example, C5a) by serine proteases of the coagulation cascade. In particular, thrombin is capable of producing C5a, which not only plays a decisive role on pathogens and infected/damaged tissues, but also acts systemically. In the case of uncontrolled complement activation, "friendly fire" is generated, resulting in the destruction of healthy host tissue. Therefore, the traditional research that focuses on a mainly positive-acting cascade has now shifted to the negative effects and how tissue damage originated by the activation of the complement can be contained. In a translational approach including structure-function relations of this ancient defense system, this review provides new insights of complement-mediated clinical relevant diseases and the development of complement modulation strategies and current research aspects.

In poly-traumatic patients a blunt chest trauma is an important trigger of the posttraumatic systemic inflammatory response. There is clinical evidence that fracture healing is delayed in such patients, however, experimental data are lacking. Therefore, we investigated the influence of a thoracic trauma on fracture healing in a rat model. Male Wistar rats received either a blunt chest trauma combined with a femur osteotomy or an isolated osteotomy. A more rigid or a more flexible external fixator was used for fracture stabilization to analyze whether the thoracic trauma influences regular healing and mechanically induced delayed bone healing differently. The blunt chest trauma induced a significant increase of IL-6 serum levels after 6 and 24 h, suggesting the induction of a systemic inflammation, whereas the isolated fracture had no effect. Under a more rigid fixation the thoracic trauma considerably impaired fracture healing after 35 days, reflected by a significantly reduced flexural rigidity (three-point-bending test), as well as a significantly diminished callus volume, moment of inertia, and relative bone surface (mCT analysis). In confirming the clinical evidence, this study reports for the first time that a blunt chest trauma considerably impaired bone healing, possibly via the interaction of the induced systemic inflammation with local inflammatory processes. ß

Background There is evidence that complement components regulate cytokine production in osteoblastic cells, induce cell migration in mesenchymal stem cells, and play a regulatory role in normal enchondral bone formation. We proved the hypothesis that complement might be involved in bone healing after fracture. Methods We investigated the expression of the key anaphylatoxin receptor C5aR during fracture healing in rats by immunostaining after 1, 3, 7, 14, and 28 days. C5aR expression was additionally analyzed in human mesenchymal stem cells (hMSC) during osteogenic differentiation, in human primary osteoblasts, and osteoclasts by reverse transcriptase polymerase chain reaction and immunostaining. Receptor functionality was proven by the migratory response of cells to C5a in a Boyden chamber. Results C5aR was expressed in a distinct spatial and temporal pattern in the fracture callus by differentiated osteoblast, chondroblast-like cells in cartilaginous regions, and osteoclasts. In vitro C5aR was expressed by osteoblasts, osteoclasts, and hMSC undergoing osteogenic differentiation. C5aR was barely expressed by undifferentiated hMSC but was significantly induced after osteogenic differentiation. C5aR activation by C5a induced strong chemotactic activity in osteoblasts, and in hMSC, which had undergone osteogenic differentiation, being abolished by a specific C5aR antagonist. In hMSC, C5a induced less migration reflecting their low level of C5aR expression. Conclusions Out in vitro and in vivo results demonstrated the presence of C5aR in bone forming osteoblasts and bone rcsorbing osteoclasts. It is suggested that C5aR might play a regulatory role in fracture healing in intramembranous and in enchondral ossification, one possible function being the regulation of cell recruitment.

Confirming clinical evidence, we recently demonstrated that a blunt chest trauma considerably impaired fracture healing in rats, possibly via the interaction of posttraumatic systemic inflammation with local healing processes, the underlying mechanisms being unknown. An important trigger of systemic inflammation is the complement system, with the potent anaphylatoxin C5a. Therefore, we investigated whether the impairment of fracture healing by a severe trauma resulted from systemically activated complement. Rats received a blunt chest trauma and a femur osteotomy stabilized with an external fixator. To inhibit the C5a dependent posttraumatic systemic inflammation, half of the rats received a C5aR-antagonist intravenously immediately and 12 h after the thoracic trauma. Compared to the controls (control peptide), the treatment with the C5aR-antagonist led to a significantly increased flexural rigidity (three-point-bending test), an improved bony bridging of the fracture gap, and a slightly larger and qualitatively improved callus (μCT, histomorphometry) after 35 days. In conclusion, immunomodulation by a C5aR-antagonist could abolish the deleterious effects of a thoracic trauma on fracture healing, possibly by influencing the function of inflammatory and bone cells locally at the fracture site. C5a could possibly represent a target to prevent delayed bone healing in patients with severe trauma.

EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3-/- and C5-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3-/-: -25%, p=0.02; C5-/-: -20% p=0.052) and newly formed bone (C3-/-: -38%, p=0.01; C5-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.

Osteoclast activity has traditionally been regarded as restricted to bone resorption but there is some evidence that also non-resorbing osteoclasts might influence osteoblast activity. The aim of the present study was to further investigate the hypothesis of an anabolic function of non-resorbing osteoclasts by investigating their capability to recruit mesenchymal stem cells (MSC) and to provoke their differentiation toward the osteogenic lineage. Bone-marrow-derived human MSC were exposed to conditioned media (CM) derived from non-resorbing osteoclast cultures, which were generated from human peripheral blood monocytes. Osteogenic marker genes (transcription factor Runx2, bone sialoprotein, alkaline phosphatase (AP), and osteopontin) were significantly increased. Osteogenic differentiation (OD) was also proved by von Kossa and AP staining occurred in the same range as in MSC cultures stimulated with osteogenic supplements. Chemotactic responses of MSC were measured with a modified Boyden chamber assay. CM from osteoclast cultures induced a strong migratory response in MSC, which was greatly reduced in the presence of an anti-human platelet-derived growth factor (PDGF) receptor beta antibody. Correspondingly, significantly increased PDGF-BB concentrations were measured in the CM using a PDGF-BB immunoassay. CM derived from mononuclear cell cultures did not provoke MSC differentiation and had a significantly lower migratory effect on MSC suggesting that the effects were specifically mediated by osteoclasts. In conclusion, it can be suggested that human non-resorbing osteoclasts induce migration and OD of MSC. While effects on MSC migration might be mainly due to PDGF-BB, the factors inducing OD remain to be elucidated.

BackgroundAfter severe blunt chest trauma, the development of an acute lung injury (ALI) is often associated with severe or even lethal complications. Especially in multiple injured patients after blunt chest trauma ALI/ARDS [acute respiratory distress syndrome (ARDS)] is frequent. However, in the initial posttraumatic phase, inflammatory clinical signs are often not apparent and underlying changes in gene-expression profile are unknown.MethodsTherefore, inflammation in lung tissue following blunt chest trauma was characterized in a well-defined bilateral lung injury model. Using DNA microarrays representing 9240 genes, the temporal sequence of blunt chest trauma-induced gene-expression patterns in lung tissue was examined.ResultsThe results suggest an activation of a highly complex transcriptional program in response to chest trauma. Chest trauma led to elevated expression levels of inflammatory and coagulatory proteins (such as TNFα receptor, IL-1α, IL-1β, C3, NF-κB and plasminogen activator). However, upregulation of proteins was found, usually incoherent of exerting effects in blunt thoracic trauma (pendrin, resistin, metallothionein and glucocorticoid-induced leucine zipper). Furthermore, significant downregulation was observed as early as 10 min after trauma for cytokines and complement factors (LCR-1, C4) as well as for intracellular signaling molecules (inhibitory protein phosphatase) and ion-channels (voltage-dependent Ca2+ channel).ConclusionsTaken together, the provided global perspective of the inflammatory response following blunt chest trauma could provide a molecular framework for future research in trauma pathophysiology.