The TLR-4-competent animals did not show an increase of serum IL-10 after the exposure of bone components to injured soft tissue as compared with control and sham animals; furthermore, serum IL-10 levels did not significantly differ between TLR-4-mutant and TLR-4-competent animals.
Pulmonary MPO activity of TLR-4-competent animals was significantly increased in the BMC, BMS, and Bone groups as compared with control and sham animals. Furthermore, sham animals showed a significant increase of pulmonary MPO activity as compared with control animals. The TLR-4-mutant animals exhibited a significantly lower pulmonary MPO activity in the BMC, BMS, and Bone groups as compared with TLR-4-competent counterparts (Fig. 3).
The BAL-serum protein ratio in TLR-4-competent animals was significantly increased in the BMC, BMS, and Bone groups as compared with the control and sham groups. The TLR-4-mutant animals showed a significantly lower protein ratio in the BMC, BMS, and Bone groups as compared with TLR-4-competent counterparts. There was no significant difference in the protein ratio between control and sham animals when comparing TLR-4-competent with TLR-4-mutant mice (Fig. 4).
Systemic inflammation and remote organ dysfunction after long bone fractures have been described in humans (1, 10, 19-21) and rodents (6, 9). However, the local mechanisms at the fracture site inducing this inflammatory cascade remain unexplained to date. It is not known whether bone components exposed to injured soft tissue are capable of inducing systemic inflammation and acute lung injury.
In our study, BMCs, BMS, and Bone all induced systemic inflammation and acute lung injury in TLR-4-competent animals when exposed to injured soft tissue. These data support our hypothesis that bone components play a pivotal role in the induction of systemic inflammation after long bone fractures via the Toll-like receptor 4 pathways.
Circulating levels of TNF-α and IL-6 are upregulated in response to injury and are established markers for systemic inflammation. Experimental and clinical data suggest that IL-6 levels correlate with the systemic inflammatory response to trauma, and that postinjury levels are predictive of systemic complications and mortality in human trauma patients (22, 23). Previous work has demonstrated increases in serum cytokines in humans and rodents after fracture (1, 6, 9, 24), hemorrhage (2, 25), and soft tissue injury (10).
In TLR-4-competent animals, all three bone components caused a significant increase in serum TNF-α levels as compared with control or sham animals. This effect was not observed in TLR-4-mutant animals. Serum IL-6 levels were significantly elevated in TLR-4-competent animals when BMC, BMS, or Bone was exposed to injured soft tissue. This effect was especially pronounced in the Bone group with even significantly higher serum IL-6 levels as compared with the BMC and BMS groups. These findings may stem from a greater proinflammatory effect of bone as compared with bone marrow. Perl et al. (26) reported finding preformed IL-6 in bone. In our study, the IL-6 concentration in the bone suspension was less than 10 pg/mL; therefore, we feel that we can exclude the injection of highly concentrated IL-6 into injured soft tissue. Serum IL-6 levels were significantly lower in the BMS and Bone groups of the TLR-4-mutant mice. However, this was not true when comparing TLR-4-competent animals of the BMC group with TLR-4-mutant animals of the BMC group. This indicates that BMCs may have low potential to activate IL-6 secretion.
The exposure of bone components to injured soft tissue caused no significant increase in serum IL-10 levels in TLR-4-competent animals. Although serum IL-10 levels are a marker for inflammation, we note that other studies also had difficulties in detecting elevated IL-10 levels after trauma. Hauser et al. (1) detected no elevated systemic IL-10 levels after fracture, and two studies reported unchanged IL-10 secretion after major surgery (27, 28). Miller-Graziano et al. (29) reported finding depressed IL-10 levels in trauma patients.
Several studies show that femur fractures are associated with a significantly increased risk of acute respiratory distress syndrome (ARDS) (5, 19, 20), and striking evidence for the importance of neutrophils in the development of ARDS is provided (30, 31). The microenvironment theory of lung injury suggests that the adherence of neutrophils to endothelium is a critical step in the development of local tissue injury (32). Recently, we were able to show that bilateral femur fracture is associated with a significant increase in pulmonary MPO activity (9). An increase in pulmonary permeability has been demonstrated in trauma patients and correlates with the development of ARDS (33). Several animal studies showed anincrease of the BAL-serum protein ratio, a marker for pulmonary permeability changes, after trauma (9, 24). In this study, TLR-4-competent animals showed pulmonary changes consistent with ARDS when bone components were locally exposed to injured soft tissue. Pulmonary MPO activity was significantly increased, and significant changes in pulmonary permeability were observed. These changes were not obvious in TLR-4-mutant mice, indicating that the local exposure of bone components induced TLR-4-dependent lung injury. The marked lung injury in TLR-4-competent animals of the BMC group was however not associated with significantly higher serum IL-6 level when compared with TLR-4-mutant mice. This finding may be explained either by our missing the serum IL-6 peak by assaying this cytokine 6 h after the exposure of BMCs to injured soft tissue in the TLR-4-competent animals, or by acute lung injury induced independently by circulating IL-6. Acute lung injury after fracture without systemic inflammation has been described in the context of fat embolism syndrome after long bone fracture (34). In our model, bone marrow-derived fat cells contained in the BMC suspension may be systemically absorbed and cause fat embolism in the lung, however, this hypothesis requires further investigation.
In addition to examining the role of specific bone components in systemic inflammation and organ dysfunction postfracture, we sought to address the role of specific signaling pathways in this process, focusing on the TLR-4 pathway. The TLR-4 has been recognized as a driver of the innate immune response in situations of sterile inflammation (35, 36) and sepsis (37-39). Several animal studies showed that systemic inflammation and remote organ dysfunction after fracture (6) or hemorrhage (11-13, 40, 41) require intact TLR-4. Levy et al. (6) showed that systemic inflammation and liver dysfunction after bilateral femur fracture were significantly reduced in TLR-4-mutant animals, and Barsness et al. (11) showed that acute lung injury after hemorrhage requires TLR-4. Our results are in accordance with previously cited studies and support our second hypothesis, namely, that systemic inflammation and acute lung injury after the exposure of bone components to injured soft tissue require an intact TLR-4 pathway. In the studies described herein, TLR-4-mutant animals did show diminished systemic inflammation and no signs of acute lung injury after the exposure of bone components to injured soft tissue. Further work is required for identifying the TLR-4-activating substances within bone and bone marrow. Previous studies have shown that both cellular and tissue matrix elements can stimulate signaling via TLR-4 (42-45). Because of the diverse composition of the exposed bone components, it seems reasonable that in this model, multiple cell types and ligands will account for TLR-4 activation.
The systemic responses observed in our study parallel those we have seen in a model of bilateral femur fracture (6, 9). Thus, for the purpose of experimental modeling, we have characterized a "pseudofracture" model. Advantages of such a model include the ability to recover these animals for longer-term studies, which is often not feasible when using fracture models.
The authors thank Lauren Kohut, Derek Barclay, and John Brumfield for technical assistance.
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