Date of Award
Doctor of Philosophy (PhD)
Dr. Jack Gauldie
Interleukin 6 (IL-6) is a pleotropic cytokine that has many important physiological roles during inflammation. One function is the regulation of the acute phase response of the liver. In order to understand the role of IL-6 as an inflammatory mediator in the acute phase response, it was necessary to study the effect that this molecule had on the regulation of its own receptors, IL-6 receptor (IL-6R, gp80 or α-receptor), and signal transducing protein, gp 130, both in vivo and in vitro.
To investigate the role that IL-6 and corticosterone play in the acute phase response and receptor regulation, we examined serum IL-6 levels, serum corticosterone levels, acute phase protein levels, and the expression of hepatic IL-6R (gp80) and gp 130 mRNA levels, in three different models of acute inflammation. Rats were treated with either Freund's complete adjuvant (FA) via intraperitoneal injection, LPS via intravenous injection, or turpentine via subcutaneous injection. All three models showed increased levels of serum IL-6 activity with LPS-treated rats inducing the quickest and greatest response (>100 ng/ml within 3h). Serum corticosterone levels increased by 3h after all treatments, and serum levels of acute phase proteins were detected within 12-24h. The expression of the IL-6R (gp80) mRNA increased as early as 3h after treatment and mRNA levels began to decline by 6-12 h. The gp 130 mRNA levels increased 2-3 fold within 24h, and the time of maximum increase differed depending on the treatment that the rat received.
Another cytokine, Leukemia Inhibitory Factor (LIF), and IL-6 share many functions in vitro, and this redundancy is thought to occur as a result of their two alpha receptors, LIF-R and lL-6R, respectively, interacting with the same signal transducing molecule, gp 130. We examined the increase of LIF-RmRNA levels in the three models of acute inflammation to determine the role of LIF during the acute phase response in comparison to IL-6. We found a maximum 2-3 fold increase in mRNA levels in comparison to controls. Maximum LIF-R mRNA levels varied depending on the type of treatment the rats received.
Overall analysis of all mRNA levels studied, showed that maximum IL-6R (gp80), gp 130, LIF-R and Cysteine Proteinase Inhibitor (CPI-an acute phase protein whose expression is regulated by IL-6) mRNA levels peaked at different times depending on the type of acute inflammation induced. Although IL-6R mRNA levels reached maximum levels quickly (3-6h) in all three models of acute inflammation, the maximum induction of gp 130 and LIF-R differed depending on the type of treatment the rats received. In all three acute inflammatory models, IL-6R and LlF-R mRNA leveIs did not peak at the same time for any of the treatments given. This suggests that although IL-6 and LIF may have similar functions in vitro, their role in vivo in inflammation is unique. Staggering of maximum LIF-R and IL-6R expression in the liver may ensure that cells are receptive to continual messages to make acute phase proteins.
To further investigate the individual effects of raised corticosterone and 1L-6 on the expression of these receptors, rats were injected with either d examethasone (DEX) or purified recombinant IL-6 (rIL-6) via intraperitoneal injection. Rats injected with rIL-6 showed a dramatic increase in both IL-6R (gp80) and gp 130 mRNA levels, as early as Ih after treatment. Dexamethasone had a significant, but less dramatic effect, on IL-6R (gp80) mRNA levels and no effect on gp 130 message. An acute phase protein response was only seen when rats were injected with rIL-6 and not DEX. Neither rIL-6 or DEX treated rats-had any effect on LIF-R mRNA levels.
To continue these studies, we investigated the long term in vivo effects of prolonged exposure to purified rIL-6 on the expression of these hepatic cytokine receptors. Repeated injections of rIL-6 did not cause decreased mRNA levels, instead increased 1L-6R (gp80), gp 130, and CPI mRNA levels (2-3 fold) were seen. Serum CPI protein levels increased gradually over a nine day period from 1.4 mg/ml to 5.8 mg/ml. LIF-R mRNA levels remained unaffected by the repeated injections of rIL-6.
The steady-state mRNA levels of the interleukin-6 receptor (IL-6R, gp80) and its signal transducing molecule, gp130, were examined in the rat hepatoma cell line, H-35, stimulated by cytokines IL-6, IL-1, Oncostatin M, and/or DEX. In contrast to our in vivo findings, in vitro DEX seemed to be the major stimulator of IL-6R mRNA expression, whereas IL-6 seemed to have little effect on the expression of its own receptor mRNA levels. However, the presence of other cytokines also influenced the corticosteroid (dexamethasone-DEX) mediated stimulation of IL-6R expression. Oncostatin M, a third cytokine related to IL-6, stimulated lL-6R mRNA levels, and this stimulation was additive with the DEX-mediated stimulation of IL-6R mRNA levels. In contrast, Interleukin 1 (IL-1) inhibited the DEX-mediated stimulation of IL-6R mRNA. At the same time, lL-1 also stimulated the presence of a second smaller mRNA transcript. This mRNA species contained the extracellular domain but lacked both the transmembrane and cytoplasmic domains of the IL-6R, suggesting alternate splicing, possibly coding for a soluble form ofgp80.
The expression of the gp130 molecule was not regulated to any major extent in vitro, and cytokines IL-6, Oncostatin M, and IL-1 all stimulated the expression of the signal transducing molecule, gp130, approximately 2 fold.
Cysteine Proteinase Inhibitor mRNA and protein levels were elevated by combinations of cytokines: IL-6+DEX, IL-6+IL-1+DEX, OSM, OSM+DEX. However, IL-1 again seemed to inhibit the IL-6+DEX mediated stimulation of CPI mRNA, possibly through inhibition of IL-6R expression and induction of a possible soluble form of the receptor.
This study shows that the combination of cytokines and hormones that interact with the hepatocyte and in turn, regulate the expression of these different receptors is very complex. In vitro, we demonstrated that both IL-1 and Oncostatin M are involved in the regulation of the IL-6 receptor complex, and that different combinations of cytokines can affect the complexity and magnitude of the hepatic acute phase response. In vivo, we demonstrated that depending on the type of acute inflammation induced, that a variety of combinations of cytokines and hormones are released, and these in tum regulate the diverse receptors on the hepatocyte, resulting in different acute phase response kinetics. Therefore, even though all of these receptors belong to a family and have similar functions, the regulation of these receptors is unique and may lead to altered cell responses in the different models of inflammation.
Geisterfer, Margit, "In Vivo and In Vitro Regulation of the IL-6 subfamily of Cytokine Receptors in the Liver" (1995). Open Access Dissertations and Theses. Paper 2251.