Detection of differentially expressed genes in particle disease using array-filter analysis.
The precise cellular mechanism of osteolysis in particle disease is still unknown. The aim of the study was to screen for new gene products in macrophages during particle contact.


METHODS
In an established macrophage model THP1-cells (human monocytic cells) were differentiated under the influence of vitamin D3 and GM-CSF into macrophage-like cells (MLC). MLCs were incubated each with different concentrations of polyethylene particles, Lipopolysaccharids (LPS) and controls. Isolated RNA was transcribed into complementary radioactive 32P labeled cDNA. This probe was hybridised on an human cDNA expression array and analysed by autoradiography. To obtain a more reliable method quantifying mRNA, the reverse transcriptase polymerase chain reaction (RT-PCR) was used.


RESULTS
The arrays showed an upregulation of the following genes by particles: TNF-Rezeptor 2, IL-1 Receptor Antagonist, Bone Morphogenic Protein 4 and HM 145. This was proven three times using RT-PCR and statistically significant in comparison to the controls. LPS induced the same upregulation except for HM145 whereas particles caused downregulation of this mRNA expression.


CONCLUSIONS
Our results prove that the model of differentiated THP-1 cells treated with PE particles is a suitable system to analyse differential gene expression patterns, since the induction of the major positive control genes TNF alpha and IL1 beta were detected by this approach. BMP 4 is known as signal protein which mediates ectopic bone formation and can also be interpreted as a contra regulatory gene. HM 145 belongs to the leukocyte chemotactic peptide receptor family. HM 145 seems to be one of the first genes that is enhanced along the septical pathway but less expressed by contact with particles. Analysis of HM 145 expression might help to diagnose septic versus aseptic loosening of prosthesis.
Rader
Ch P
ChP
Department of Orthopadics, University of Wuerzburg.
Baumann
B
B
Rolf
O
O
Radke
S
S
Hendrich
Ch
Ch
Schütze
N
N
Seufert
J
J
Jakob
F
F
D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D005809:Genes, Regulator; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D008264:Macrophages / Q000378:metabolism*; D008854:Microscopy, Electron; D020411:Oligonucleotide Array Sequence Analysis; D010014:Osteolysis / Q000235:genetics* / Q000473:pathology; D020959:Polyethylene; D011475:Prosthesis Failure; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000235:genetics; D047992:TNF Receptor-Associated Factor 2
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D016207:Cytokines; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D011506:Proteins; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D047992:TNF Receptor-Associated Factor 2; D020959:Polyethylene