459

Cytokine Differences in Mature and Immature Human Macrophage Cell Lines J.L. Morgan*, R.E. Singer
Procter & Gamble Co., Mason, OH, USA
ABSTRACT
The goal of this investigation was to contrast the cytokine of macrophages within inflamed gingival tissues may response profiles following LPS activation of mature and influence the balance of the host response to LPS challenge.
PGE2 Time course: HL-60 cells #2
immature human macrophage phenotypes in order to assess the possibility that differences in macrophage maturity might OBJECTIVE
have impact on the clinical outcomes following macrophage recruitment and activation in inflamed gingival tissues. Human macrophage cell lines previously characterized as The goal of this investigation was to contrast the cytokine promyelocytic (HL-60) and the more mature (THP-1) cells response profiles following LPS activation of mature and were cultured and used to assess the effects of LPS immature human macrophage phenotypes. The outcome challenge. Following differentiation with PMA both cell lines would provide information as to whether differences in were chal enged with E.coli LPS and culture supernatants macrophage maturity might impact clinical outcomes were collected and analyzed for TNF-alpha , GM-CSF , following macrophage recruitment and activation in inflamed Time Course: THP-1 cells #2
PGE2 and PGF2 alpha levels at various time points. HL-60 gingival tissues. cells secreted TNF-alpha with a peak level achieved 6-8 hours following activation with LPS. HL-60 cells secreted MATERIALS AND METHODS
very low levels of GM-CSF following 6-8 hours of incubation which increased only modestly before peaking at levels substantially less than that of TNF-alpha 24-48 hours after THP-1 cells are a human, nonadherent, monocytic cell line LPS addition. The mature macrophage cell line THP-1 purchased from the American Type Culture Collection peaked in production of TNF-alpha 6-8 hours following LPS (ATCC). HL-60 cells, a promyelocytic cell line (immature) challenge; however, THP-1 production of GM-CSF was were also obtained from ATCC; passages 20-40 were used substantial 6 hours following LPS-mediated activation and in these experiments. Both cell lines were passaged three PGE2 levels are low in HL-60 and markedly higher in THP-1 cells. increased markedly for 24-48 hours to levels to exceeding times per week in RPMI 1640 (GIBCO), supplemented with
that of TNF-alpha. PGE2 and PGF2 alpha were both 10% heat-inactivated fetal bovine serum (Hyclone) and
significantly higher in the THP-1 cells than in the HL-60 cells. penicillin (100U/ml) and streptomycin (100 ug/ml).
These findings demonstrate that the kinetics of the
expression of the expression of these cytokines in THP-1 cells (ATCC) were differentiated by exposure to 16nM
macrophages differ markedly and that production of GM- Phorbol Myristate Acetate (PMA) for 18 hrs. Cells were
CSF is substantially greater in the more mature THP-1 cell pelleted by centrifugation, counted, and viability determined.
line. Since TNF-alpha and GM-CSF mediate very different Cells were seeded into 24-well plates at 5 x105 cells/well/ml.
host responses and PGE2 is known to suppress TNF-
alpha , these findings suggest that the maturity of HL-60 cells were pelleted, counted, and viability determined.
macrophages infiltrating inflamed gingival tissues might These cells were seeded at 1.5 x 106 cells/well/2ml. directly
have an important impact on the balance of the host into 24-well plates and differentiated by exposure to 32nM
response (inflammatory vs. protective) to LPS PMA for 24 hrs. The adherent cells were washed and
challenge.
checked for viability before beginning the time course. INTRODUCTION
For both cell lines, each time point had unchallenged control wells (no LPS) and LPS- challenged wells at 1 ug/ml. Cell Bacterial components such as LPS (lipopolysaccharide) are supernatants were removed at the various time points and able to activate macrophages to synthesize and secrete frozen at -70° C. Analyses for TNF and GM-CSF were many different inflammatory mediators that affect gum performed using R&D Systems ELISA kits. Eicosanoid disease, such as TNF and PGE assays were performed using Cayman EIA kits. Research presented at the 76th General Session of the IADR; Nice, France June 24-27, 1998 Cytokine Differences in Mature and Immature Human Macrophage Cell Lines J.L. Morgan*, R.E. Singer
Procter & Gamble Co., Mason, OH, USA
RESULTS (cont.)
Time course: HL-60 cells #1
CONCLUSION
LPS challenge 1 ug/ml
-TNF and GM-CSF have very different kinetics. -TNF peaks early in both cell types but is directionally higher in THP-1 cells. -GM-CSF production is delayed in both cell lines but is ~ 10x higher in THP-1 cells. -PGE2 production parallels GM-CSF and is much higher in THP-1 cells. -These findings suggest that maturity of macrophages Time course: THP-1 cells #1
impacts the balance of the cytokine responses: more LPS challenge at 1 ug/ml
mature cells secrete higher levels of GM-CSF and PGE2. Time course: HL-60 cells #2
LPS challenge 1 ug/ml
Time course: THP-1 cells #2
LPS challenge at 1 ug/ml
Research presented at the 76th General Session of the IADR; Nice, France June 24-27, 1998

Source: http://www.dentalcare.be/media/fr-BE/journals/pgresrch/posters/iadr98/pdfs/iadr98pp_1814.pdf