total joint replacement by endoprosthesis is a widely used procedure to restore functionality of joints in patients with osteoarthritis . although surgery is usually safe and successful , complications may arise , particularly due to bacterial infections on and around the implant . according to the literature , the risk of infection is approximately 1 to 2% in primary arthroplasty [ 13 ] . considering the ever - increasing number of prostheses which are implanted every year , infections result in high socioeconomic costs since the treatment is often prolonged and expensive . the common cause of implant - associated infection is the formation of bacterial biofilms on the implant . first , bacteria adhere to foreign surfaces , and then they produce and embed themselves in an extracellular substance , the name - giving  film .  among others , bacteria in biofilms acquire a relative resistance towards many antibiotics [ 6 , 7 ] ( reviewed in [ 6 , 812 ] ) . therefore , extensive antibiotic treatment often fails , which makes revision surgery with tissue debridement mandatory . because bone infections are associated with loss of bone substance ; loosening of implants is a common complication and requires an exchange of the prosthesis . however , each revision surgery bears an increased risk of yet another infection . in implant infection , staphylococci species are prevalent , but other species including enterococci or streptococci are also found and possibly also infections with multiple species [ 12 , 13 ] . bacterial biofilms elicit a profound local immune response with infiltration of neutrophils , monocytes , and t cells , associated with the local generation of proinflammatory cytokines [ 14 , 15 ] . basically , neutrophils are able to attack and destroy biofilms [ 1619 ] , but in the case of implant infections , host - defence mechanisms may be inefficient and a persistent and progressive inflammatory response might ensue , causing tissue damage and bone resorption ( osteolysis ) . presumably , the cytokine - rich proinflammatory environment promotes the generation of bone resorbing osteoclasts from myeloid precursor cells , but the exact mechanisms are still unclear . in this context , the possible participation of the macrophage inflammatory proteins mip1 ( ccl3 ) and mip2 ( cxcl2 ) was assessed in this study . their participation in host defence against infection and in acute or chronic phases of inflammation is well documented . there is , however , increasing evidence for production of these cytokines by cells other than monocytes , macrophages , or neutrophils , for example , by endothelial cells , fibroblasts , neural tissue , and a variety of tumor cells ( for review see [ 2026 ] ) . both , ccl3 and cxcl2 , are also described in the context of osteoclast generation and osteolysis , particularly in the mouse [ 2729 ] . to assess their participation in implant - associated infection , we analysed gene expression and protein expression in biopsies derived from patients with implant - associated infection and for comparison in patients with aseptic loosening . the latter is an example for a sterile inflammation , which presumably is caused by the uptake of implant - derived wear particles by phagocytic cells and which also eventually leads to implant loosening . furthermore , in a series of vitro experiments , primary osteoblasts as a possible source of ccl3 and cxcl2 were assessed , as well as their role in the induction of osteoclastogenesis . patients who underwent revision surgery due to a prosthetic infection and patients suffering from aseptic loosening of a total joint replacement ( articulating materials either metal - on - polyethylene or ceramic - on - polyethylene ) were included in the study . diagnosis of loosening was based on patients ' complaints , clinical examination , and examination by conventional x - ray and/or ct scan . from five patients with an infection and five patients with an aseptic loosening of an implant , the samples were immediately placed into rna later ( ambion life technologies , darmstadt , germany ) for quantitative pcr analysis . immediately before surgery , blood samples from patients with implant - associated osteomyelitis ( n = 39 ) , aseptic loosening ( n = 22 ) , or healthy donors ( n = 10 ) were collected for gene expression analysis and elisa ( see below ) . the tissue specimens were formalin - fixed , decalcified in ethylenediaminetetraacetic acid ( edta ) , and paraffin embedded . the biopsies were examined , the diagnosis of an acute or chronic osteomyelitis was made , and the cellular infiltrates in particular the pmn , lymphocytes , and monocytes / macrophages were quantified . the paraffin - embedded tissue sections ( 3 - 4  m ) were also used for immunohistochemical analyses . prior to antibody incubation , heat pretreatment in citrate buffer ( ph 6.1 ) was performed . as primary antibody , the monoclonal mouse anti mip-1alpha was used ( r&d systems , darmstadt , germany ) . from cells , mrna was isolated with the magnapure - lc device using the mrna - i standard protocol . tissue samples were disrupted with ribolyser devices ( thermohybaid , heidelberg , germany ) containing 400  l lysis buffer from the magnapure mrna isolation kit containing 1% dtt ( v / w ) ( roche diagnostics , mannheim , germany ) . mrna was isolated with the magnapure - lc device using the mrna - standard protocol for cells . mrna was reversely transcribed using amv - rt and oligo-(dt ) as primer ( first strand cdna synthesis kit , roche , mannheim , germany ) according to the manufactures protocol . primer sets optimized for the lightcycler ( ras , mannheim , germany ) were purchased from search - lc gmbh ( http://www.search-lc.com/ ) . the pcr was performed with the lightcycler faststart dna sybr greeni kit ( ras ) according to the protocol provided . to control for specificity , the copy number was calculated from a standard curve , obtained by plotting known input concentrations of four different plasmids at log dilutions to the pcr - cycle number ( cp ) at which the detected fluorescence intensity reaches a fixed value . to correct for differences in the mrna content , the transcript numbers were normalized according to expression of the housekeeping gene peptidylprolyl isomerase b ( ppib ) . staphylococcus aureus ( seattle 1945 , atcc 25923 , wesel , germany ) and staphylococcus epidermidis ( rp62a , atcc 35984 , wesel , germany ) were grown overnight on a blood agar plate at 37c ( number pb5039a , thermo scientific , germany , wesel ) . the following day , two to three colonies were picked from the agar plate and placed in phosphate buffered saline and adjusted to 1  10 cells / ml . monocytes were isolated from the peripheral blood of healthy donors ( informed consent was obtained and the institutional guidelines were observed ) . the blood was layered on polymorphprep ( axis shield , oslo , norway ) , and the monocyte fraction was recovered . monocytes were positively selected using anti - cd14 micro beads ( miltenyi biotec , bergisch gladbach , germany ) . the cells were seeded into 24-well dishes ( nunctm , wiesbaden , germany ) at a concentration of 1  10 cells / ml in rpmi containing 10% fetal calf serum , 1% glutamine , and 1% penicillin / streptomycin ( medium and supplements were obtained from gibco life technologies , darmstadt , germany ) . primary osteoblast cultures derived from bone marrow of patients undergoing autologous bone graft harvesting using the reamer - irrigator - aspirator ( ria ) technique   were cultivated in osteoblast growth medium ( promocell , heidelberg , germany ) containing 0.1% penicillin / streptomycin ( gibco life technologies ) . cells were subcultivated following digestion with trypsin ( 0.05% trypsin - edta , life technologies , uk ) for 5 minutes at 37c and resuspended in osteoblast growth medium . after 2 days , homogenous cell layers were seen ; osteoblasts were identified by expression of collagen type i and lack of markers for myeloid cells . , osteoblasts were seeded into 24-well dishes ( nunctm , wiesbaden , germany ) at a concentration of 2  10 cells / ml osteoblast growth medium . for the stimulation assays , 1  10 monocytes or 2  10 osteoblasts were each placed in 1  ml medium ( rpmi or osteoblast growth medium , resp . ) into a 24-well plate . bacteria ( s. aureus or s. epidermidis ) were added in a ratio of 20 , 100 , or 500 bacteria per osteoblast and incubated for 2 hours at either 4c or 37c . the cells were washed twice with phosphate buffered saline ; then , 400  g of vancomycin was added in 1  ml culture medium for 30 minutes at 37c . the medium was replaced by fresh medium containing 20  g vancomycin , and cells were incubated for 24 hours or 48 hours . for stimulation with lta , osteoblasts or monocytes were incubated with lta ( sigma , munich , germany ) for 24 to 48  h. all experiments were carried out in duplicates and repeated at least three times with cells of different patients . ccl3 and cxcl2 in cell culture supernatants and in serum samples were determined using commercially available elisa kits according to the protocol provided by the manufacturer . the human cxcl2 elisa kit was purchased from hlzel diagnostika ( cologne , germany ) , the human ccl3 kit from r&d systems ( minneapolis , usa ) . cd14 + monocytes were seeded into 24-well dishes ( nunctm , wiesbaden , germany ) at a concentration of 1  10 cells per well and allowed to adhere for 24 hours in medium ( rpmi , gibco life technologies , darmstadt , germany ) . then , nonadherent cells were removed by washing , and the remaining cells were incubated in rpmi supplemented with 10% fcs ( pantm biotech , aidenbach , germany ) , 1% penicillin / streptomycin , 1% glutamine ( all obtained from gibco ) , and m - csf ( 25  ng / ml ; r&d systems , minneapolis , usa ) . for stimulation rankl ( 50  ng / ml , peprotech , hamburg , germany ) was added or ccl3 ( mip1 ) ( r&d systems , minneapolis , usa ) in different concentrations . cultures were incubated at 37c in 5% co2 for up to 14 days with a change of medium and removal of nonadherent cells at day 7 . for the followup differentiation , the cells were fixed with 4% pfa for 15 minutes at 37c and identified by their morphological appearance as giant cells with multiple nuclei . binding of fitc - labeled phalloidin ( sigma - aldrich ; 1  :  20 dilution for 40 minutes ) revealed the typical actin ring formation . nuclei were stained using dapi ( invitrogen , oregon , usa ) , ( diluted 1  :  30000 for 5 minutes ) . additionally , staining with an antibody against cathepsin k was performed ( anti - cathepsin k ( santa cruz biotechnology , heidelberg , germany ) was diluted 1  :  50 ) , and detected with anti - mouse igg - cy3 . fluorescence was visualized using a digital fluorescence microscope ( keyence , neu - isenburg , germany ) . for quantification , cells were counted on images of six high - power fields and the percentage of osteoclasts in relation to the total cell number was calculated . following stimulation with ccl3 or rankl ( santa cruz biotechnology , heidelberg , germany ) , cells ( 3  10 ) were lysed with ripa buffer ( santa cruz biotechnology , santa cruz , usa ) and mixed with sds - gel sample buffer and separated by sds - polyacrylamide gel ( 12% ) electrophoresis , followed by blotting to a pvdf membrane ( millipore , eschborn , germany ) . the antibody to nfatc1 was purchased from santa cruz biotechnology ( heidelberg , germany ) , and as secondary antibody peroxidase - labelled anti - mouse igg ( jackson immuno research , pennsylvania , usa ) was used . for detection , amersham ecl plus western blotting detection system ( ge healthcare limited differences between groups were calculated using mann - whitney test using origin 9.0 software . significance level was determined as p < 0.05 . gene expression varied among the patients , but , in all , expression was higher in tissue from osteolytic sites compared to muscle . cxcl2 expression was also higher in patients with infection compared to patients with aseptic loosening , whereas expression of ccl3 did not differ between patients with infection and those with aseptic loosening ( data summarised in figure 1 ) . immunohistological examination confirmed the presence of ccl3 positive inflammatory cells particularly neutrophils , lymphocytes , and monocyte / macrophages . osteoclasts were seen in the proximity of ccl3 positive inflammatory cells in the eroded bone ( an example for a patient with infection and osteolysis is shown in figure 2 ) . by analysing gene expression of cd14 , infiltration of mononuclear cells into affected sites could be confirmed , with higher expression of cd14 at osteolytic sites compared to muscle ( 2132.9  1487.9 cd14 gene transcripts versus 864.6  512.5 in muscle in patients with infection ; 671.7  479.2 versus 316.6  189.9 in patients with aseptic loosening ) and higher expression in patients with infection compared to tissue from aseptic loosening ( figure 1 ) . expression of cathepsin k , a marker for osteoclasts and hence of osteolysis , did not differ between the two patient groups ( figure 1 ) . in the peripheral blood , ccl3 was found in 8 of 14 patients with aseptic loosening ( 51.5 to 92.8  pg / ml ) and in 5 of 34 patients with an infection ( 47.5 to 118.8  pg / ml ) ; cxcl2 was not detected in sera of patients with aseptic loosening and in 5 of 34 patients with an infection ( ranging from 9.4 to 514.5  pg / ml ) . of note , these were not the same patients as those with measurable ccl3 serum concentrations . in serum of healthy donors neither ccl3 nor cxcl2 gene expression of cxcl2 and ccl3 in peripheral blood cells did not differ between the two patient groups , and peripheral blood cells responded essentially similar to stimulation with either pma or lps ( figures 1(e ) , and 1(f ) ) . infiltrating monocytes are a likely source of cytokines , but also osteoblasts are known to produce and release cytokines in response to infectious agents ( [ 34 , 35 ] ) . to follow up on that issue , primary osteoblasts were cocultivated with lta as an example for a bacteria - derived entity . within 2 to 6 hours , an increase in gene expression of ccl3 and cxcl2 bacteria were then killed and cell culture was continued for 24 hours or 48 hours . mips were released into the supernatant depending on the number of bacteria present ( representative experiment in figure 3(b ) ) . on average , s. epidermidis when added in a relation of 500 bacteria / cell induced cxcl-2 production of 22014.75  12815.2  pg / ml , s.aureus 13966  6128.36  pg / ml . for unstimulated cells , 6608.2    3412.2 pg / ml was determined ( values are the mean  sd of three experiments with cells derived from different patients ) . ccl3 release was only seen with 500 s. epidermidis / cell and amounted to 71.44    27.3  pg / ml versus 0 in unstimulated cells . s. aureus  in similar bacteria to cell ratio  did not elicit a ccl3 response . of note , when osteoblasts were exposed to the bacteria at 4c as opposed to 37c , ccl3 and cxcl2 release was within the same range ( data not shown ) . also , there was no significant difference between cultivation for 24  h and 48  h. as expected , monocytes released both , ccl3 and cxcl2 , into the supernatant . both bacterial strains were equally efficient ( on average 317385  pg / ml of ccl3 and 31890  pg / ml of cxcl2 were released ) and again there was no major difference between 37 and 4c ( data not shown ) . to link generation of mips to osteolysis , the effect of ccl3 on the differentiation of monocytes towards osteoclasts was assessed . after 14 to 18 days in culture , giant cells with multiple nuclei appeared , expressing cathepsin k and trap , enzymes characteristic of osteoclasts ( example in figure 4(a ) ) . because osteoclast generation depends on an extended activation and de novo synthesis of nfatc1 [ 36 , 37 ] , lysates of ccl3 treated monocytes were analysed by western blotting . enhancement of nfatc1 was seen , though to a lesser extent when compared to rankl - treated cells ( figure 4(b ) ) . tissue samples of patients with implant - associated infections revealed gene expression of ccl3 and cxcl2 at sites of osteolysis , exceeding the amount detected in unaffected muscle tissue . expression of cd14 , a marker of infiltrating mononuclear cells , followed essentially the same pattern , suggesting that in accordance with data in the literature ccl3 and cxcl2 are predominately produced by macrophages ( reviewed in ) . in patients with aseptic loosening basically a similar expression pattern of cxcl2 , ccl3 , and cd14 was seen , though transcript numbers for cxcl2 and cd14 were lower compared to those seen in infected tissue . aseptic loosening is a sterile inflammation presumably caused by prosthesis - derived wear particles which activate primarily myeloid cells [ 30 , 38 , 39 ] . the presence of very potent stimulators of myeloid cells in infectious cases ( particularly bacteria - derived lipopolysaccharides or lipoteichoic acid ) could explain the more prominent reaction seen in bacterial infection . moreover , neutrophils , which are found primarily in infected but rarely in aseptic tissue , could contribute to ccl3 and cxcl2 production and cause a more pronounced inflammatory response . differences in the extent of the inflammatory response could explain why aseptic loosening usually progresses slower in some patients , 10 to 20 years after primary arthroplasty . eventually , however , the extent of bone degradation is rather similar , compatible with our data , showing similar transcripts for cathepsin k , a parameter for osteoclast generation . the fact that cxcl2 gene expression in infected tissue was enhanced compared to tissue of patients with aseptic loosening , but that of ccl3 was not , suggests that its production is differently regulated or involves cells others than macrophages . indeed , ccl3 , for example , is also produced by a subset of cd8 + t cells [ 40 , 41 ] , or endothelial cells . fibroblasts produce both , ccl3 and cxcl2 , , as do neutrophils   after stimulation . important for our studies was the possible production of these cytokines by osteoblasts [ 28 , 45 ] , a fact we were able to confirm by exposing cultivated osteoblasts to staphylococci or lipoteichoic acid ( lta ) , a major component of the bacterial cell wall . lta stimulated both , ccl3 and cxcl2 expression , whereas the bacteria induced predominantly cxcl2 , suggesting different signalling pathways . lta most likely activates the osteoblasts via toll - like receptor 2 , whereas staphylococci express in addition to lta also other surface molecules which may mediate binding and activation of target cells . among those is protein a on s. aureus which binds to the tumor necrosis factor  receptor on osteoblasts [ 46 , 47 ] . others include fibronectin - binding proteins which are expressed by bacteria and mediate binding to surface - associated fibronectin on osteoblasts [ 4850 ] . because s. aureus uses a different binding site on fibronectin as s. epidermidis , different signalling pathways are triggered , which could explain that synthesis of cxcl2 is stimulated by s. aureus , but that of ccl3 is not . activation of cxcl2 synthesis occurred even when the osteoblasts were exposed to bacteria at 4c , indicating that the mere contact of bacteria to cell surface receptors is sufficient to trigger the cells and ruling out phagocytosis as a crucial signal . enhanced local cytokine production can result in enhanced serum concentrations , and serum cytokines can serve as biomarkers to monitor disease activity . elevated serum concentrations of mips were found in various chronic inflammatory diseases , including rheumatoid arthritis or systemic lupus erythematosus [ 5155 ] , as well as in experimentally induced infection in mice [ 56 , 57 ] . differentiating between aseptic implant loosening and implant failure due to an infection can be difficult , because the standard laboratory parameters for infection ( serum crp concentration or leukocyte count ) can be negative in infectious cases and thus misleading . taking the different therapeutic approaches in aseptic and infectious cases into consideration , the search for a reliable preoperative marker has become crucial in orthopaedic surgery . however , neither the ccl3 and cxcl2 protein concentrations in serum , nor the gene expression by peripheral blood cells differed between aseptic and infectious cases . most likely , in contrast to the above mentioned systemic diseases , the expression and effects of ccl3 and cxcl2 are exhibited exclusively locally , with no  spill - over  into the peripheral blood . locally generated ccl3 and cxcl2 could contribute as chemoattractants to the infiltration of cells ; cxcl2 is a major chemokine for neutrophils , which could account for the neutrophil infiltration into infected tissues since expression of cxcl2 is enhanced in these patients as opposed to aseptic cases ; ccl3 attracts predominantly cd8 + t cells , in line with the shift of the cd4/cd8 ratio towards cd8 + in the cellular infiltrate in implant - associated infection . aside from attracting immune cells , cxcl2 and ccl3 could have an additional function in implant loosening . mips have been shown to participate in osteoclast generation in patients with multiple myeloma and the expression correlated with bone degradation [ 5961 ] . moreover , in vitro studies and various rodent models provided proof of a connection between mip - production and bone degradation [ 6265 ] . in a series of in vitro studies , we were now able to show that ccl3 induced the differentiation of cd14 + monocytes to osteoclasts , which might be relevant for bone degradation in osteomyelitis , because peripheral blood monocytes are known to be osteoclast precursors . osteoclast differentiation induced by ccl3 was associated with nfatc1 synthesis , in line with the effect of rankl , a well - known and major inducer of osteoclastogenesis [ 67 , 68 ] . under our experimental conditions , ccl3 was less effective when compared to rankl , which , however , not necessarily diminishes their role in osteoclast generation in vivo . the relevance of ccl3 and cxcl2 in the process of bone resorption was shown in mouse experimental models . in conclusion , our study demonstrates that , under infectious conditions , but also in sterile inflammation , cxcl2 and ccl3 are produced locally . because mips may contribute to osteoclast generation , these findings provide a link between local inflammation and bone resorption which further leads to implant loosening . colocalisation with cd14 suggests synthesis by cells of the monocyte / macrophage lineage , but , because osteoblasts can also produce mips when appropriately stimulated , it is feasible that they initiate the inflammatory response by recruiting leukocytes . this in turn creates a proinflammatory environment which results in a progressive course of the disease and leads to extensive tissue damage , as well as loss of bone .