INTRODUCTION
Post-traumatic osteomyelitis after osteosynthesis or of total joint replacement represents one of the major complications of orthopedic and trauma surgery. Posttraumatic osteomyelitis is defined as a device-associated infection, resulting in progressive inflammatory disease, which in the majority of patients requires removal of the implant and even extensive reconstructive surgery due to tissue destruction and osteolysis (1–5).
In most patients bacteria, predominantly staphylococci, can be isolated from the infected site (1–6). In line with an infectious etiology, osteomyelitis occurs more frequently in patients with open fractures or extensive soft tissue injury, in immunocompromised patients, and in patients with diabetes mellitus and complications of wound healing (reviewed in Refs. 1–5).
Although an infectious etiology of post-traumatic osteomyelitis is largely accepted, a number of other factors are discussed that could contribute to the development of disease or to persistence of an infection, including pre-existing diseases, surgical procedures, and properties of the implant, the latter in part learned from experimental studies (reviewed in Refs. 1–5).
The generation on implants of bacterial biofilms could provide an explanation for persistent infection: bacteria organized in biofilms are resistant towards antibiotics and most probably also towards host defense mechanisms. By releasing toxins and probably also proteolytic enzymes, the persisting bacteria could contribute to tissue destruction (7–12).
As an alternative mechanism for tissue destruction we propose infiltration into the infected site of leukocytes, which because of their proinflammatory, cytotoxic, and collagenolytic potential could cause tissue destruction (13). Indeed, in the present study, we found a prominent leukocyte infiltrate around the infected implants. The infiltrate consisted predominantly of highly activated polymorphonuclear neutrophils (PMNs), expressing surface receptor patterns similar to those of PMNs exposed to bacterial products in vitro.
MATERIAL AND METHODS
Patients
After having obtained informed consent and after approval by the local ethic committee, 18 patients with post-traumatic osteomyelitis were recruited into a prospective study between December 2001 and January 2003. The diagnosis was based on the medical history, the clinical examination, x-rays, as well as increased leukocyte count and C-reactive protein levels. All patients required surgery with removal of the implant or the endoprosthesis. The patients differed with regard to age, sex, localization of the primary implant/endoprosthesis, and the interval between the primary implantation and the development of osteomyelitis (data summarized in Table 1). From all patients swabs were taken from the site of inflammation. In 14 of 18 patients bacteria could be cultivated and identified (Table 1).
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Table 1: Clinical and laboratory data of the patients
Recovery of the local immunocompetent cells
During surgery the tissue surrounding the infected implant/endoprosthesis was rinsed with 50 to 100 mL of sterile pyrogene-free 0.9% saline using a 50 mL syringe. The so-called “lavage” was collected in sterile tubes containing heparin (2500 IU per 50 mL) and sodium azide (final concentration 0.1%). The samples were processed within 3 h. The cells were collected by centrifugation, washed with sterile phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin and 0.1% sodium azide (FACS-puffer), and subjected to cytofluorometry (see below). Viability was assessed by trypan-blue exclusion and microscopic evaluation; >90% of cells were viable.
Peripheral blood
From the patients peripheral venous blood (5 mL) was taken immediately before surgery and 5 to 7 days postoperatively. For comparison, blood of healthy donors (n = 20) was taken. Cells in whole blood were subjected to cytofluorometry as described below.
Cytofluorometry
For cytofluorometry in whole blood double-labeling was performed using a fluorescein isothiocyanate (FITC)-labeled antibody to CD66b (former CD67) as a PMN-marker and phycoerythrine (PE)-labeled murine monoclonal antibodies to either CD14, CD16, CD18, CD19, CD34, CD62L, and CD64. All antibodies were obtained from Immunotech (Marseille, France) as were isotypic mouse IgG1 FITC and IgG2a PE. The final concentration of antibodies varied between 1–20 μg/mL whole blood.
After incubation of whole blood with the respective antibodies, erythrocytes were lyzed using FACS-lysing solution provided by Becton and Dickinson (Heidelberg, Germany). Cells were analyzed by FACSCalibur® and CellQuest® software (Becton and Dickinson). Results are expressed as % positive cells in the respective gate or quadrant, or as mean fluorescence intensity (MFI), respectively.
Uptake of lipoteichoic acid (LTA) by PMNs
LTA (from Staphylococcus aureus, purchased from Sigma, München, Germany) was labeled with FITC using 5-(and-6)-carboxyfluorescein, succinimidyl ester (5(6)FAM, SE) purchased from Molecular Probes (Eugene, OR) strictly following the protocol supplied by the manufacturer. To study uptake, 10 μg of LTAFITC was mixed with 100 μL of heparinized blood. After incubation at 37°C for various times; the cells were washed with PBS containing 1% sodium azide. To detect CD14 and CD18 on the same cells, PE-labeled antibodies were used (see above). The erythrocytes were then lyzed using FACS-lysing solution (Becton and Dickinson). The remaining leukocytes were suspended in PBS containing 1% paraformaldehyde and subjected to cytofluorometry.
Activation of PMNs in whole blood by LTA
One milliliter of whole blood from healthy donors was incubated with LTA in various concentrations and for various times at 37°C. Thereafter, expression of surface receptors was measured by cytofluorometry as described above. To analyze the signaling pathways, the following inhibitors were used: cytochalasin B, cycloheximide 1-pyrrolidinecarbodithioic acid (PDTC) as an inhibitor of NF-κB activation (14) (all obtained from Sigma), and SB203580 (Calbiochem, Marburg), an inhibitor of the p38 mitogen-activated protein (MAP) kinase. The inhibitors were added to the blood before exposure to LTA. The concentrations used did not affect the viability of the PMN as measured by propidium iodide and trypan blue exclusion, respectively.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) for detection of CD14 specific mRNA
Total RNA was isolated from 1 × 107 PMN after 1 and 24 h of culture with the LTA (1 μg). RNA isolation was performed using a RNAeasy kit by Quiagen (Hilden, Germany) strictly following the instructions of the manufacturer. RNA was transcribed using the 1st Strand cDNA synthesis kit for RT-PCR (AMV, Boehringer, Mannheim, Germany). RT-PCR was performed according to the method described for monocytes in (15). The RT products were separated by a 1.5% agarose gel, stained with SybrGreen (Molecular Probes, Leiden, Netherlands) and analyzed by FLA2000 (Fuji, Japan) using Image Gauge V3.0 (Fuji) as software. Marker: Boehringer DNA Marker VI (range 154–2176 bp) in a concentration of 30 μg/ml. β actin was used as housekeeping gene as described in (16).
RESULTS
Patient characteristics and clinical course
The study comprises 18 patients with posttraumatic osteomyelitis after infection of osteosynthesis implants or of endoprostheses. The patients varied with regard to age, sex, type and localization of the implant, as well as the time interval between implantation and the development of osteomyelitis. The clinical and laboratory data of the patients are summarized in Table 1. In some patients profound osteolysis proximate to the implant was seen on the x-rays (examples are shown in Fig. 1A and B).
Fig. 1: Osteolysis documented by x-rays. A, A total joint replacement of the left knee was performed in July 1999 (patient 11); in February 2001, the endoprosthesis was still in a correct position without any signs of osteolysis; evidence for osteolysis became apparent in August with osteolysis progressing further (gray arrows) and resulting in instability, pain, and local signs of inflammation. In February 2002, the endoprosthesis had to be removed (upper panel: lateral view; lower panel: anteroposterial view). B, Osteolysis around the nail and the screws (arrows) is shown in patient 18 after re-osteosynthesis with a reamed nail (November 2001). Reosteosynthesis was performed because the patient had developed a pseudarthrosis of the tibia after a previous callus distraction, which was required because of a chronic osteitis after a comminuted fracture of the tibia.
In all patients, removal of the implant or the endoprosthesis was required, permitting analysis of the local infection, and even more importantly, of the local inflammatory response. In 14 patients bacteria, mainly gram-positive species, could be identified from locally taken swabs (Table 1).
Analysis of leukocyte infiltrates
Before, during, and after removal of the infected implant or the endoprosthesis, respectively, the inflamed site was rinsed with sterile saline and recovered as “lavage fluid.” Aside from erythrocytes, this lavage fluid contained between 1 to 2 × 107 viable leukocytes, indicating cellular infiltration into the infected site. The majority of these cells (75–90%) were identified as polymorphonuclear neutrophils (PMN) positive for CD66b. T-lymphocytes, identified by CD3 expression, amounted to 5–15%. B-lymphocytes (CD19 positive), monocytes (CD14 positive, CD66b negative), and stem cells (CD34 positive) were rarely seen (an example is shown in Fig. 2; data of all patients are summarized in Table 2).
Table 2: Identification of infiltrated leukocytes
Fig. 2: Leukocytes recovered from the lavage fluid (data shown for patient 1) and visualized by cytofluorometry. By forward-side scatter analysis (left) two major populations were seen (R1 and R2), which by monoclonal antibodies were identified as PMN (R1: CD66b positive; filled peak) and T-cells (R2: CD3 positive; filled peak; the thin lines represent the respective isotype controls).
To analyze the infiltrated PMN further, expression of surface receptors, known to be associated with bacterial defense, was studied. CD14, the receptor for lipopolysaccharides (LPS) or LTA, which is only weakly expressed on PMN of healthy donors, was drastically upregulated on the patients' PMN. Of note is, that in all patients the infiltrated PMN expressed more CD14 than PMN derived from the peripheral blood of the same patient taken immediately prior to surgery (an example is shown in Fig. 3; data of all patients are summarized in Fig. 4).
Fig. 3: Expression of CD14, CD18, CD62L, and CD64 on PMNs of the peripheral blood of a healthy donor (upper panel); peripheral blood of a patient with posttraumatic osteomyelitis (middle panel); and PMNs derived from the lavage of the same patient (lower panel). The thick lines show the respective antibody binding; the thin lines the isotype controls.
Fig. 4: Expression of CD14, CD18, CD62L, and CD64 on PMNs of the peripheral blood of healthy donors (n = 20; open boxes); of the peripheral blood of the patients (striped boxes); and of the lavage fluid (black boxes). The boxes contain 50% of the values. The difference between the groups was calculated using the t test for unpaired samples (n.s. differences are not significant; P > 0.05).
The β2 integrin CD18, which is constitutively expressed on PMN, was upregulated on both, the PMN of the lavage fluid and of the peripheral blood, but more prominent on the latter (Figs. 3 and 4).
Another receptor weakly expressed on PMN of healthy donors is the high affinity receptor for IgG (FcγI; CD64). Upregulation was seen on the infiltrated PMN and to a lesser extent on peripheral PMN of the patients (Figs. 3 and 4). Expression of the leukocyte selectin CD62L, which is constitutively expressed by PMN, was severely reduced in the infiltrated cells, whereas the levels on PMN of the peripheral blood were near normal (Figs. 3 and 4).
CD16, the low-affinity receptor for IgG (FγIII), was measured in 11 of the 18 patients. A more complex expression pattern was seen: in the peripheral blood of three patients expression was within the range of healthy donors; in 4 patients enhanced expression was seen and in four others a loss of CD16. In the infiltrate, expression was normal or marginally reduced (data not shown).
The differences seen between the infiltrated PMN recovered from the lavage and the PMNs derived from the peripheral blood suggests a predominant local activation. Accordingly, there was no correlation between surface alterations of the lavage-derived PMNs and the elevation of systemic inflammatory markers, such as C-reactive protein (CRP) or the leukocyte count. In PMN of the peripheral blood CD14 expression was significantly higher in patients with high CRP levels (> 20 mg/L) compared with patients with lower CRP levels; a similar trend was seen for CD64 expression (Fig. 5). That the upregulation of CD14, CD18, and CD64 coincided with infection was seen in follow-up studies: after surgery with removal of the implant the surface receptor expression approached normal values in all patients within 1 to 4 weeks. Furthermore, in patients with reduced CD16 expression normal levels were seen after recovery (data not shown).
Fig. 5: Association of CRP levels with expression of CD14 (left panel) or CD64 (right panel) on PMNs derived from the peripheral blood (left) or the lavage fluid (right). The open boxes contain data from patients with CRP levels lower than 20 mg/L; the filled boxes with CRP higher 20 mg/L. An association with CRP levels was only seen for cells of the peripheral blood.
Effect of LTA on PMN receptor expression in vitro
To investigate how the surface receptor expression of PMNs can be modulated, a set of in vitro experiments was performed using PMNs of healthy donors and LTA, a constituent of the bacterial wall of gram-positive bacteria. Because it is known that activation of PMNs varies widely between individuals, all experiments were performed with blood of at least three different donors. To closer reproduce the in vivo situation, whole blood samples were incubated with LTA for various times. With use of FITC-labeled LTA uptake over time by PMN could be demonstrated (Fig. 6). Of note is, that binding of LTA increased for up to 2 h, concomitantly with upregulation of CD14 and CD18.
Fig. 6: Binding of FITC-labeled lipoteichoic acid to PMNs of a healthy donor. A–D show the controls: isotype controls (A); expression of CD14 on PMN (identified by CD66b; B); expression of CD18 (C); and LTA-FITC, all measured at time 0 (D). E, Over time (10 min; 30 min; 60 min; 90 min), an increase of LTA-FITC binding was measured simultaneously with upregulation of CD18 or CD14, respectively. (The isotype controls did not change with time; these data are not shown.)
CD14, which is only found in low copy numbers on PMN of healthy donors, increased further over time for up to 48 h (an example is shown in Fig. 7A; data of different experiments using cells from 3 different donors are summarized in 7B). The upregulation within the first 2 h could be inhibited by cytochalasin B, a blocker of exocytosis, indicating release of the preformed protein. Further upregulation of CD14 could be inhibited by cycloheximide, indicating dependence on de novo protein synthesis. In line with this finding in response to LTA CD14-specific mRNA was induced within 1 h (Fig. 7D). To assess the signaling pathways specific inhibitors of the p38 MAP kinase and of NF-κB activation were used. With both inhibitors, upregulation of CD14 could be blocked (data are summarized in Fig. 7C). In all experiments viability of PMN was not affected by the respective inhibitors as tested by trypan blue exclusion (data not shown).
Fig. 7: Upregulation of CD14 on PMN of healthy donors exposed to LTA. A, By cytofluorometry, a time-dependent upregulation of CD14 was seen (data from one of 12 different donors are shown). Whole blood (100 μL) was incubated with LTA (1 μg) for the times indicated. B, data of four different donors are shown (closed circles: exposed to LTA; open circles: incubated without LTA; data are presented as mean ± SD). * indicates values different between LTA-stimulated and unstimulated cells. The interindividual differences account for the large standard deviation. C, The presence of the NF-κB inhibitor PDTC (10 nM) or the MAP kinase inhibitor SB203580 (100 μM) during the cultivation of PMN with LTA (1μg/106 PMN) for 24 h greatly reduced upregulation of CD14 (one of three experiments using PMN of three different donors is shown). D, By RT-PCR an increase in abundance of CD14 transcripts was seen after incubation with LTA. The abundance of β actin, which was used as housekeeping gene, did not change (one of three experiments using PMN of three different donors is shown).
CD64 expression increased rapidly after exposure to LTA and reached plateau after 1 h. Then, expression persisted for the next 48 h (Fig. 8). De novo protein synthesis was not required (data not shown). CD18 initially increased within 1 h; however, by prolonged culture, a decrease to baseline value was seen (an example is shown in Fig. 8). CD16 expression was upregulated within the first 20 min; thereafter a decline was seen, which by 12 h was well below baseline value and decreased even further by 24 to 48 h (Fig. 8). CD62L declined progressively over time. By 60 min 50% of the CD62L was lost (Fig. 8).
Fig. 8: Alteration of CD16, CD18, CD62L, and CD64 expression on PMN of healthy donors exposed to LTA. The left panels show the alteration in cells of a single donor over time measured by cytofluorometry; the right panels show the accumulated data of three different donors (closed circles: exposed to LTA 1 μg/100 μL blood). * indicates that the values are different from controls (open circles: nonstimulated cells) and from baseline values; ** different from the baseline value only.
DISCUSSION
Our data derived from 18 patients with posttraumatic osteomyelitis clearly demonstrate the presence of leukocytes in the tissue surrounding the implant, indicating that the leukocytes had infiltrated the infected site. This observation is in line with previous histological studies by others (17). The infiltrate consisted predominantly of PMNs, which represent the first line defense against bacterial infections and are the major effector cells of the innate immunity and of the acute inflammatory response as well (18,19).
The infiltrated PMNs were highly activated as indicated by a change of the surface receptor pattern. Based on numerous in vivo and in vitro studies alteration of surface receptors are reliable markers of PMN activation (16,20–25).
The differences in the receptor patterns between PMNs derived from the peripheral blood of healthy donors with that of the patients and with the PMNs recovered from the infected tissue lead to the following conclusions: PMNs are already activated in the peripheral blood, most probably by bacteria-elicited mediators. Most prominently here is the upregulation of CD18, an adhesion protein required for the emigration of the PMNs from the blood vessel to the infected site. Once there, expression of CD18 returns to baseline values. The selectin CD62L, which is also necessary for the emigration of PMNs, also is down-regulated on the locally recovered cells, whereas the receptors for recognizing bacteria, CD14 and CD64, are particularly high on the infiltrated PMN. The infiltrated PMN have a prolonged life-span as seen by upregulation of CD14, which requires de novo protein synthesis, and by expression of CD16, the low affinity receptor for IgG, which is only lost when PMN undergo apoptosis (26,27). Taken together, these data indicate a further, local activation of the PMN in the infected tissue.
That alteration of surface expression was linked to inflammation became evident from the follow-up studies: on peripheral blood PMN receptor expression approached normal levels after surgery, indicative of a causal relationship between infection and alteration of surface receptor patterns.
Infiltration into infected sites is the pathophysiological relevant function of PMN, and is essential for an efficient host defense. Key elements are generation or release of reactive oxygen species; of NO and a number of cytotoxic and bactericidal entities. Because these agents are also toxic for the surrounding tissue, the acute PMN response has to be limited in a time- and spatial fashion (19,28,29). This is normally achieved by monocytes/macrophages which within days or even hours infiltrate the infected site to clear up the exhausted and by then apoptotic PMN; the uptake by macrophages prevents release of cytotoxic and proteolytic mediators from PMN, thereby limiting tissue destruction (30,31).
Contrary to the normal situation, in posttraumatic osteomyelitis, the PMNs persist at the site of infection; they are not apoptotic, but rather highly activated and there is no evidence for monocytes infiltration. Consequently, the destructive potential of PMNs remains unrestrained and could inflict tissue damage, including osteolysis, because of the release of proteolytic and collagenolytic enzymes.
To gain insight into the molecular mechanisms of PMN survival and receptor alteration, in parallel studies PMN of healthy donors were cultivated under infection analogue conditions. In place of intact bacteria, LTA as a major cell wall component of gram-positive bacteria was used. We now could demonstrate, that LTA binds to and activates PMN: a rapid upregulation of CD14, CD16, CD18, CD64, and loss of CD62L were seen; prolonged culture resulted in a further upregulation of CD14 caused by de novo synthesis, as the experiments with the protein synthesis inhibitors indicate. As seen for monocytes, LTA signaling involves the MAP kinase and NF-κB activation (32,33).
The high CD64 expression persisted for up to 48 h. After 3 to 12 h CD18 decreased to baseline values and CD16 even further. Thus, prolonged culture generated an activated phenotype, CD14high, CD64high, and CD18near normal, and CD62Llow and depending on the time point CD16 normal or low closely resembling the PMN of the cellular infiltrates. Loss of CD16 is associated with cell death by apoptosis, at least under culture conditions (26,27). That in the majority of patients PMN express normal-to-high levels of CD16 underlines the fact that the PMNs were viable.
That PMNs recovered from the infected site were still viable, indicates that they had escaped, at least temporarily, their constitutive or their phagocytosis-induced apoptosis (34–36). Thus, it is feasible that also in posttraumatic osteomyelitis the infiltrated PMNs escape from apoptosis due to the continuous activation by not yet defined mediators (37). Alternatively, the lack of apoptosis-inducing signals generated during phagocytosis could account for the longevity of PMNs (38).
Inadequate phagocytosis could also explain why despite the presence of activated PMNs, living bacteria could still be recovered from the same site. Different bacteria species were identified; so it is unlikely that the relative resistance to phagocytosis was due to bacteria-inherent properties. Rather, we presume the generation of bacterial biofilms on the implants, which confers protection to host defense mechanisms. The existence of biofilms on materials used for implants or endoprostheses, respectively, was described, as well as biofilm formation as the cause for persistent infections (6,8,9,39).
Organization in biofilms, however, does not prevent interactions of the PMN with the bacteria. Recent data indicate that PMN adhere to biofilms and might also get activated (40). On one hand, activation might result in the release of toxic and proteolytic entities from the PMN. On the other hand, recognition of bacterial surface structures, such as LTA, might also activate the phagocytic capacity of the PMN. In the attempt to phagocytose the biofilm, the PMN release cytotoxic mediators and proteolytic enzymes, a phenomenon known as “frustrated phagocytosis” (41). We propose that in posttraumatic osteomyelitis persistent PMN activation in concert with “frustrated phagocytosis” and the lack of regulatory monocytes cause tissue degradation and eventually osteolysis.
ACKNOWLEDGMENTS
The skillful technical assistance of Birgit Denefleh and Friederike Hug is greatly appreciated.
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