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Kobbe, Philipp*; Kaczorowski, David J.; Vodovotz, Yoram†‡; Tzioupis, Christopher H.*; Mollen, Kevin P.; Billiar, Timothy R.; Pape, Hans-Christoph*

doi: 10.1097/SHK.0b013e31816f257e
Basic Science Aspects

Remote and systemic inflammatory responses after long bone fractures have been well described, but the mechanisms underlying these changes remain unexplained. We hypothesized that bone components locally exposed to injured soft tissue are capable of inducing a systemic inflammatory response associated with acute lung injury, and that this inflammatory cascade requires Toll-like receptor 4 (TLR-4) signaling. Accordingly, male C3H/HeOuJ (TLR-4-competent) and C3H/HeJ (TLR-4-mutant) mice were injected with various bone components (bone marrow cells, bone marrow supernatant, and bone suspension, respectively) in bilaterally injured thigh muscles and euthanized after 6 h. Serum TNF-α, IL-6, and IL-10 levels, and pulmonary myeloperoxidase activity was measured using specific enzyme-linked immunosorbent assay kits. Pulmonary permeability changes were assessed with bronchoalveolar lavage. Local exposure of bone components to injured soft tissue induced systemic inflammation and acute lung injury in TLR-4-competent, but not in TLR-4-mutant, animals. These findings suggest that bone components contribute to systemic inflammation and acute lung injury after long bone fractures via TLR-4 signaling and support the notion of a central role for TLR-4 in sensing tissue damage.

*Departments of Orthopaedic Surgery and Surgery, and Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

Received 31 Dec 2007; first review completed 11 Jan 2008; accepted in final form 12 Feb 2008

Address reprint requests to Philipp Kobbe, MD, Department of Orthopaedic Surgery, University of Pittsburgh, Kaufmann Medical Bldg, Suite 1010, 3471 Fifth Ave, Pittsburgh, PA 15213. E-mail:

This study was supported by the National Institutes of Health Trauma Center (grant no. P50-GM053789). Kevin P. Mollen is a recipient of an American College of Surgeons Resident Scholarship.

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Traumatic injury may lead to both local and systemic inflammation (1, 2). Systemic inflammation can in turn lead to remote organ dysfunction, which, despite continuous improvement in intensive care medicine, still represents a major cause of death in the trauma population (3, 4). Long bone fractures are a major contributor to post-traumatic systemic inflammation and acute lung injury (5-10). The underlying link between long bone fractures and the systemic inflammatory response is not well understood. Specifically, the contribution of components of fractured bone to the systemic inflammatory response is not known. Recently, it has been shown that Toll-like receptor 4 (TLR-4) signaling is activated after tissue injury with the induction of a sterile inflammatory cascade. Systemic inflammation with consequent liver dysfunction after bilateral femur fracture (6) and hemorrhage-induced remote organ dysfunction require TLR-4 signaling (11-13).

Bone and bone marrow cells present immunologic properties that may play an important role in the induction of acute systemic inflammation after long bone fractures. The local cascades depicting the interplay between bone/marrow and immune cells have been previously described for physiological (bone healing) and chronic pathological (osteoarthritis, osteoporosis) processes (14-18). We hypothesized that local exposure of bone components to injured soft tissue contributes to systemic inflammation and acute lung injury after long bone fracture. Furthermore, we hypothesized that this systemic response requires intact TLR-4 signaling. Our findings suggest that the local exposure of bone components to injured soft tissue is sufficient to initiate a cascade of systemic inflammation associated with acute lung injury, and that these responses are diminished in TLR-4-mutant animals.

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Animal care

This research protocol complied with the regulations regarding the care and use of experimental animals published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Pittsburgh. Male C3H/HeOuJ and C3H/HeJ mice (Jackson Laboratories, Bar Harbor, Maine), aged 6 to 10 weeks and weighing 20 to 30 g, were used in the experiment. C3H/HeOuJ are TLR-4-competent mice, whereas C3H/HeJ mice have a point mutation in the intracellular region of the TLR-4 gene resulting in the generation of a dominant negative allele with consequent defects in TLR-4 signaling. The animals were maintained in the University of Pittsburgh Animal Research Center with a 12:12-h light-dark cycle and free access to standard laboratory food and water.

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Harvesting of bone components

Either C3H/HeOuJ or C3H/HeJ mice were euthanized, and the long bones of the lower extremities were harvested under sterile conditions. To obtain approximately equal concentrations, two femurs and two tibias were used for the following protocol: the bone cavities of the long bones were opened, and the bone marrow was flushed out with a total of 0.5 mL of phosphate buffer solution (PBS) using a 27-gauge needle. The bone marrow was then flushed through a 70-µm cell strainer (BD Biosciences, Bedford, Mass), followed by separation of cellular and noncellular bone marrow components by centrifugation at 1,200 rpm for 10 min. The cellular bone marrow components were resuspended in 0.3 mL of PBS. The remaining bone cortices were crushed with a mortar and pestle and suspended in 0.5 mL of PBS. This suspension was then homogenized and flushed through a 70-µm cell strainer (BD Biosciences).

This procedure supplied three bone components: bone marrow cells (BMCs), bone marrow supernatant (BMS), and bone suspension (Bone). Cell count of the BMC suspension showed 60 to 80 × 106 nucleated cells per 1 mL.

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C3H/HeOuJ and C3H/HeJ mice were divided into five subgroups, respectively. Animals were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg) and inhaled isoflurane (Abbott Laboratories, Chicago, Ill). In the control group, animals were euthanized after the induction of anesthesia to obtain physiological baseline levels. All other animals were subjected to minimal soft tissue injury by crushing both thigh muscles with a clamp for 30 s, followed by the sterile injection of 0.1 mL of either PBS (sham group), BMCs (BMC group), BMS (BMS group), or bone suspension (Bone group) into each injured thigh muscle using a 27-gauge needle. Animals were euthanized 6 h after the injection. To prevent a graft-versus-host reaction, bone components were not mixed between the two animal strains.

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Blood collection for serum cytokine assessment

After thoracotomy, cardiac blood was withdrawn under deep anesthesia during exsanguination for euthanasia. Blood samples were allowed to clot at 4°C. After 24 h, blood samples were centrifuged at 7,000 rpm for 7 min to separate the serum from cellular and clotting components. Serum was stored at −20°C until thawed for TNF-α, IL-6, and IL-10 quantification with enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, Minn).

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Bronchoalveolar lavage

The trachea was cannulated, and the lungs were lavaged twice with a total of 1.5 mL of PBS, which was instilled and then slowly withdrawn over 30 s. The recovered bronchoalveolar lavage (BAL) fluid (greater than 80% of that delivered) was centrifuged at 1,200 rpm for 10 min, and the supernatant was frozen at -90°C until further analyzed. Bronchoalveolar lavage protein concentrations were quantified with the BCA Protein Assay (Pierce, Rockford, Ill) and were normalized to serum protein levels (BAL-serum protein ratio).

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Lung myeloperoxidase activity

To minimize background myeloperoxidase (MPO) activity by remaining nonadherent intravascular polymorphonucleated cells, a needle was inserted into the beating right ventricle after cardiac blood withdrawal, and the pulmonary circulation was perfused with 1.5 mL of PBS. After BAL, the lung was harvested and immediately snap frozen in liquid nitrogen and stored at -90°C. To determine lung MPO activity, lung tissue samples were thawed and homogenized in a lysis buffer exactly as directed by the manufacturer. Lung MPO activity was measured using an MPO-enzyme-linked immunosorbent assay kit (Cell Sciences, Canton, Mass) and normalized to the protein concentration of the sample (BCA Protein Assay Kit, Pierce).

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Statistical analysis

Results are expressed as means ± SEM of 4 to 6 animals per group. Subgroup comparisons of the TLR-4-competent animals were assessed using the Kruskal-Wallis test followed by the Mann-Whitney U rank sum test. Group comparisons between TLR-4-competent and -mutant mice were assessed by the Mann-Whitney U rank sum test. The null hypothesis was rejected for P < 0.05. The data were analyzed using StatView Version 5.0 (SAS Institute, Cary, NC).

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Serum cytokine levels

The TLR-4-competent animals of the BMC, BMS, and Bone groups exhibited significantly higher serum TNF-α levels as compared with control and sham animals. The TLR-4-mutant mice of the BMC, BMS, and Bone groups demonstrated significantly lower levels of circulating TNF-α as compared with their TLR-4-competent counterparts. There was no significant difference in serum TNF-α between control and sham animals when comparing TLR-4-competent with TLR-4-mutant mice (Fig. 1). In comparison with the control and sham groups, TLR-4-competent animals of the BMC, BMS, and Bone groups exhibited significantly increased serum IL-6 levels. Furthermore, serum IL-6 levels were significantly increased in the Bone group as compared with the BMC and BMS groups. Serum IL-6 levels of the BMS and Bone group in TLR-4-mutant animals were significantly lower than in their TLR-4-competent counterparts. There was no significant difference in serum IL-6 levels between control, sham, and BMC animals when comparing TLR-4-competent with TLR-4-mutant mice (Fig. 2).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

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.

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Lung injury

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).

Fig. 3

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).

Fig. 4

Fig. 4

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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.

Limitations of this pseudofracture model are that only the quantity of injected BMCs was quantifiable, whereas the concentration of BMS and bone in each suspension is unknown. To minimize these effects, we standardized the amount of long bones and PBS used to harvest the bone components.

We conclude that both bone and bone marrow components exposed to injured soft tissue induce TLR-4-dependent systemic inflammation and acute lung injury, which may in part explain the sustained systemic response to long bone fractures. However, other contributors to this highly complex immune response, such as soft tissue injury, fracture hematoma, and input from the central nervous system, have to be recognized. Furthermore, our data suggest that strategies directed at the level of the TLR-4 receptor may mitigate fracture-induced inflammation and remote organ dysfunction.

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The authors thank Lauren Kohut, Derek Barclay, and John Brumfield for technical assistance.

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Long bone fracture; bone marrow; soft tissue injury; multiple organ failure; pseudofracture model; Toll-like receptor 4

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