Abstract
Cultured cells depend on cytokine mediators for sustained growth and maintenance and are routinely employed in bioassays to detect and measure minute changes in biological mediators, e.g. the interferons and interleukins. We evaluated the effects of mycoplasma infection on the steady-state mRNA levels of two cytokines IL-1 alpha and beta. Noninfected human squamous carcinoma cell line A431 expressed constitutively IL-1 alpha and beta mRNA. In contrast freshly isolated peripheral blood mononuclear cells and the monocytic cell line U937 expressed abundant IL-1 mRNA only after the appropriate stimulation. Peripheral blood mononuclear cells and U937 steady-state IL-1 beta mRNA levels were considerably greater than IL-1 alpha mRNA levels, whereas nearly equivalent high levels of IL-1 alpha and beta mRNA were detected in A431 cells. Mycoplasma infection of cultured A431 cells reduced the steady-state levels of IL-1 alpha and beta mRNA. This effect was nonspecific for A431 cells as actin mRNA steady-state levels showed similar decreases to mycoplasma contamination. However, this response was cell specific since mycoplasma-free and contaminated U937 cells differed little in IL-1 mRNA expression. These results show that the response to mycoplasma infection is at least partly cell-type dependent.
Highlights
EXPERIMENTAL PROCEDURESThe costs of publication of this Human Peripheral Blood Mononuclear Cells-Six separate monoarticle were defrayed in part by the payment of page charges
From the Department of Medicine, Divisionof Immunology and Allergy, University of Geneva, Hospital Cantonal, CH-1211Geneva 4, Switzerland and the $Department of Microbiology, University Medical Schoolof Geneva, Switzerland
We have employed cDNAprobes to human interleukin-la and /3 (IL-la and p), corresponding to PI forms 5.0 and 6.8, to evaluate whether mycoplasma infection influences the expression of IL-1 mRNA under in vitro culture conditions
Summary
The costs of publication of this Human Peripheral Blood Mononuclear Cells-Six separate monoarticle were defrayed in part by the payment of page charges. The cDNA probes cillin, and 100 pg/ml streptomycin and in thepresence (24 h) or not to mRNAs for IL-la and@T,NF (Marmenout et al, 1985),and total of12.5 pg/mlConA, 15 ng/ml phorbol ester-l@-phorbolester-4@- human actin (Gunning et al, 1983) and a 900-base pairs fragment phorbol 12 myristic 13-acetate (PMA),or for 12 h with 3 pg/ml from the 23 S rRNA gene of Mycoplasmahyorhinis PSP64-129 (Gobel lipopolysaccharide,and 2 p~ calcium ionophore (A21387).The mon- and Stanbridge, 1984) were nick-translated to a specific activity ocytic lymphoma cell line U937was first cultured for 3 days of greater than 10' dpm/pg and employed at 1.5-3.0 X 10' dpm/ml differentiation in RPMI-1640,10% fetalcalf serum, 2 mM glutamine, hybridization solution. A431 and U937 cells were washed 2 times in cold phosphate-buffered saline, quenched in liquid nitrogen for RNA extraction, or extracted for cell-associated IL-1 activity
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