Detection and modulation of interleukin-1 receptor antagonist messenger ribonucleic acid and immunoreactivity in Graves' orbital fibroblasts.
OBJECTIVE
To analyze the expression and regulation of intracellular and soluble interleukin-1 (IL-1) receptor antagonist (IL-1RA) in orbital fibroblasts and to determine whether a disbalance between IL-1 receptor agonist and antagonist may promote IL-1 mediated proinflammatory actions in Graves' ophthalmopathy (GO).


METHODS
Early passages of cultured orbital fibroblasts (OFs) derived from four patients with active GO and six control subjects were examined for their baseline expression of the two variants of IL-1RA, intracellular IL-1RA (icIL-1RA) and soluble IL-1ra (sIL-1RA. In addition, modulation of icIL-1RA and sIL-1RA was studied after exposure of OF to a broad range of cytokines as well as to nonspecific stimulating agents such as lipopolysaccharide and phorbol-12-myristate 13-acetate. The IL-1RA gene and protein variants were analyzed by reverse transcriptase-polymerase chain reaction, immunocytochemical staining, immunoblotting, and enzyme-linked immunosorbent assay.


RESULTS
At baseline, both GO- and control OF showed low levels of constitutive icIL-1RA ribonucleic acid and absence of sIL-1RA ribonucleic acid expression. Exposure to various cytokines stimulated icIL-1RA and sIL-1RA gene expression in both groups, but generally to markedly lower levels in GO-OF compared to that of control OF (P < 0.01). Analysis of IL-1RA protein expression showed low levels of constitutive IL-1RA immunoreactivity (22 kDa) in cell lysates and absence of sIL-1RA immunoreactivity in culture supernatants derived from both GO-OF and control OF. Interleukin-1-alpha was capable of inducing expression of two variants (23 and 26 kDa) of IL-1RA immunoreactivity in supernatants, derived from control OF, and to a lesser degree, GO-OF (P < 0.01). Quantitative analysis showed markedly lower abundance of IL-1RA immunoreactivity in cell lysates and supernatants derived from GO-OF monolayers compared to that detected in control OF (P < 0.001).


CONCLUSIONS
Our results demonstrate differences in regulation of icIL-1RA and sIL-1RA and markedly lower levels of expression in cultured GO-OF compared to normal OF. Failure to generate, upon cytokine stimulation, sufficient quantities of icIL-1RA and sIL-1RA to balance agonist stimulation of the IL-1 receptor may facilitate IL-1-dependent proinflammatory and fibrogenic actions within the orbital tissue in GO.
Mühlberg
T
T
Department of Internal Medicine, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany.
Heberling
H J
HJ
Joba
W
W
Schworm
H D
HD
Heufelder
A E
AE
D018929:Cell Culture Techniques; D016207:Cytokines / Q000494:pharmacology; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D006111:Graves Disease / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D053590:Interleukin 1 Receptor Antagonist Protein; D008070:Lipopolysaccharides / Q000494:pharmacology; D009915:Orbit / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D012795:Sialoglycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016207:Cytokines; D017931:DNA Primers; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; D012795:Sialoglycoproteins; D013755:Tetradecanoylphorbol Acetate