Is And Brain Illp

As already noted, the ICV administration of IL-1 produces behavioral, endocrine, physiological, and neurochemical sequelae that resemble those induced by stressors, as well as the acute phase responses produced by IS described above (DeSimoni, Seroni, DeLuigi, Manfridi, Mantovani, & Ghezzi, 1990; Morimoto, Sakata, Watanabe, & Murakami, 1989). It is therefore of interest to determine whether IS might induce IL-lp in the brain. This issue is complex for a variety of reasons. One is that adrenal corticosteroids (CORT) destabilize IL-lp mRNA (Amano, Lee, & Allison, 1993) and inhibit IL-lp gene transcription (Lee, Tsou., Chan, Thomas, Petrie, Eugui, & Allison, 1988) as well as a number of translational and p os I-translation a 1 processes (Kern, Lamb, Reed,Daniele, & Nowell, 1988) involved in the production of IL-1 p protein (see Watkins, Nguyen, & Maier this volume). Because IS increases adrenal CORT levels, we sought to determine whether IS would increase brain IL-ip protein levels in both adrenalectomized and sham surgery subjects. Basal CORT was maintained at normal levels by the addition of CORT to the drinking water. Thus the ADX subjects had normal basal CORT and a normal circadian rhythm of CORT (the rats drink much more during the dark part of the cycle), but could not increase CORT in response to IS.

IL-ip protein levels were measured by ELISA. Several methodological issues should be noted at the outset. The procedure involves administration of IS or control treatment, sacrifice, dissection of brain into regions, and separation of protein from brain by sonication and centrifugation. It is therefore possible that any or all of the detected IL-ip derives from IL-lp circulating in the cerebral vasculature or bound to IL-1 receptors on the inside of the vascular endothelium. To assess this possibility comparisons between IL-1 p levels measured in saline-perfused and non-perfused brains have been compared, and the resulting data do not differ. Data to be described were obtained following perfusion. Second, ELISA uses an antibody (Ab) specifically directed against rat IL-ip to detect IL-1. However, there is always the possibility that the Ab that is used is capable of detecting another protein as well. We have used a number of approaches in an attempt to overcome this potential interpretive difficulty, a) Three different Abs directed against rat IL-lp have been used. Although absolute values have differed between Abs, the direction and magnitude of the changes have been the same for all Abs. b) Specificity of Ab binding was assessed by Western blot analysis. Strong bands appeared at approximately 17kDa, the molecular weight of mature (active) IL-1 p. However, faint bands were also detected at roughly 33kDa, the molecular weight of the precursor (inactive) form of IL-1 p. Several laboratories have now reported that Ab directed against 17kDa IL-lp recognizes 33kDa pro-IL-ip with approximately 10-fold less affinity, likely due to a conformational change conferred by amino acid sequences present in pro-, but not active IL-lp (Dinarello, 1992). c) Total protein from the brain sonication supernatants was seperated on a Sephadex G-50 column, and fractions assayed with the ELISA. The signal was restricted to the fraction in the molecular weight range of IL-lp. d) The rat IL-ip Ab was verified for its ability to recognize pure rat IL-ip by immunoprecipitation. Details of these Ab verification procedures can be found in Nguyen et al. (1998).

Fig. 4 shows IL-ip protein levels in hypothalamus and hippocampus, while Fig. 5 shows data for the nucleus tractus solitarius and pituitary gland. Samples were taken either immediately, 2hr, 24 hr, or 48 hr after IS or home cage control treatment in adrenalectomized (ADX) and sham surgery subjects.. The general pattern is that IL-lp protein levels are elevated immediately and 2hr after IS in adrenalectomized subjects. Although appearing small in the figure because of the scale used, IL-ip increased by 2-3 fold in the hypothalamus immediately and 2hr after IS in sham subjects. It should be noted that the increase in IL-lp was regionally specific and failed to occur in other regions tested. For example, IL-1 levels did not increase in posterior cortex, in either sham or adrenalectomized subjects.

The fact that brain IL-lp protein increases were detectable immediately after the 2hr IS session deserves comment. It is not yet known whether IS increases mRNA for IL-ip, nor at what point during the stress session that this might occur. IL-lp gene transcription and translation have been reported to occur as rapidly as within 1 hr (Abel & Czop, 1992), and mRNA increases have been observed within 15min of stimulation in vitro and in vivo (Enk, Angeloni, Udey, & Katz, 1993). Thus the IL-lp observed could reflect newly synthesized protein. Alternatively, the IL-lp measured could be the result of post-translational processing of preformed IL-ip precursor. As noted above, IL-lp is synthesized as a 33kDa precursor (pro-IL-lp) that is biologically inactive, with mature IL-ip being formed from pro-IL-ip by proteolytic cleavage.

Timepoint of IL-1 after IS

Hypothalamus

Hypothalamus

SHAM ADX-cort

Hippocampus

Sham ADX-cort

Figure 4. IL-1 p protein levels measured in hypothalamus and hippocampus of sham (SHAM) or adrena-lectomized (ADX) rats given either inescapable shock (IS) or home cage control treatment (HCC). Samples were taken either immediately, 2 hr, 24 hr, or 48 hr after treatment.

Sham ADX-cort

Figure 4. IL-1 p protein levels measured in hypothalamus and hippocampus of sham (SHAM) or adrena-lectomized (ADX) rats given either inescapable shock (IS) or home cage control treatment (HCC). Samples were taken either immediately, 2 hr, 24 hr, or 48 hr after treatment.

The proteolytic processing is accomplished by IL-1 converting enzyme (ICE), and ICE activity can be regulated in a number of different ways (Keane, Giegel, Lipinski, Callahan, & Shivers, 1995). There is evidence that there is constitutive expression of preformed pro-IL-ip in the brain, and in the hypothalamus in particular (Tringali et al. 1996). The active forms of ICE also result from proteolytic cleavage from an ICE precursor, and stimulation by LPS has been shown to produce the rapid processing of ICE, leading to the production of mature IL-1 p. It is possible that IS also increases ICE activity in this fashion, thereby leading to the rapid production of mature IL-1 p from existing pro-IL-ip. Interestingly, ATP induces a rapid increase in ICE activity and IL-lp processing such that mature IL-lp is released within 7.5 min of ATP administration (Griffiths, Stam, Downs, & Otterness, 1995), and IS is known to increase ATP levels (Minor & Saade. 1997). Furthermore, neurons that utilize ATP as a transmitter project from the A1 cell group via the ventral noradrenergic bundle to the hypothalamus (Sperlagh, Sershen, Lojtha, & Vizi, 1998), the region in which IL-1 increases after IS are most readily detectable.

Timepoint of 1L-1 after IS

Nucleus Tractus Solitarius

Nucleus Tractus Solitarius

Sham

ADX-cort

Sham

ADX-cort

Pituitary

Pituitary

2 hr 24 hr

Defeat Depression

Defeat Depression

Learning About How To Defeat Depression Can Have Amazing Benefits For Your Life And Success! Discover ways to cope with depression and melancholic tendencies! Depression and anxiety particularly have become so prevalent that it’s exceedingly common for individuals to be taking medication for one or even both of these mood disorders.

Get My Free Ebook


Post a comment