Metodología de los análisis

T h e response to inflammation in humans occurs in three phases (for review see Fey and Fuller, 1987); immediate local responses such as vasodilatation and release of lysosomal enzymes; secondary responses such as neutrophil chemotaxis and thirdly systemic reactions including pain, fever and a rise in the plasma concentration of certain proteins collectively known as the acute phase reactants. A common characteristic of these proteins is their involvement in defence mechanisms against tissue dam age, infection or bleeding. Fibrinogen is one such protein. It is important that there is regulation of the response so that it gradually becomes quiescent and homeostasis is restored, otherwise significant tissue dam age can occur._...

Interleukin-6 (IL-6) is the major regulator of the acute phase response (Castell et al,1 989) and monocytes, macrophages, fibroblasts and endothelial cells are probably the major sources of IL-6 in the inflammatory state. The liver is the major target organ in vivo, although other cell types in transformed cell lines have shown som e response in vitro (Baumann et al,1986). Hepatocytes have a specific IL6 receptor on their surface which comprises two proteins, one of which binds IL6 and through interaction with the second, a trans-mem brane tyrosine kinase (Kishimoto et al,1992), stimulates the phosphorylation of specific cytoplasmic proteins. This initiates a cellular cascade of events which results amongst other things in the rapid modification of a nuclear transcription factor NF-IL6, which

significantly enhances the DNA-binding ability of this protein (Akira et al, 1992). N F -IL6 is a ‘leucine-zipper* containing protein which has homology to the transcription factor C A T enhancer binding protein (C/EBP). The transcription of a number of liver specific genes are controlled by C /EB P binding, due to the presence of a sequence elem ent (consensus TG TG G A A A ) in the promoter region of both positive and negative acute phase genes; such an elem ent is found in both the promoters of the negative acute phase proteins, albumin and apoAI. It appears that NF-IL6 competes for C /EB P binding in these genes and this has the effect of suppressing the transcription of negative acute phase proteins. By contrast, positive acute phase proteins have related sequence elements which are recognised only by NF-IL6 and binding results in strong transcription; such elem ents have been identified in the fibrinogen gene promoter amongst others. IL-6 induces a liver-specific nuclear protein to bind to the promoter region of the acute phase genes (Poli and Cortese,1989). Dalmon and coworkers (1993 ) have shown that in the R-fibrinogen gene promoter, there are three subdomains in the region of the IL-6 responsive element (IL-6R E) and that all three are needed for full response to IL-6. Apart from the IL-6RE itself, the other two domains are the hepatic nuclear factor 1 (H N F 1) binding site, which is both the major determinant of and essential for R-fibrinogen gene tissue specific expression, and a second site which binds several distinct nuclear proteins from the C /E B P family and plays an important role in the constitutive expression of the gene. These three domains lie in the region -70bp to -160bp from the start of transcription (Dalmon et al, 1993).

In addition to IL-6, glucocorticoids are important mediators of the acute phase response and experiments examining their effects are of interest. Baumann and coworkers (1990) showed in HepG2 cells that dexam ethasone, a powerful synthetic glucocorticoid, increased the transcriptional activity of several acute phase proteins including fibrinogen and a^-acid glycoprotein. The response for fibrinogen w as specific to the HepG 2 cells, whereas that for the a^-acid glycoprotein also occured in mouse L-cells. Experiments using rat hepatocytes and the FAZA rat hepatoma cell line showed that IL-6 increased m R N A production of the fibrinogen genes resulting in a 1.5-3 fold rise in fibrinogen protein levels (Otto et al, 1987). Dexam ethasone alone increased the fibrinogen protein levels to the sam e degree but did not increase m RNA levels. The dexam ethasone effect was blocked in the FAZA cells by cyclpheximide showing that protein synthesis w as required for maximum transcription to occur. Using IL-6 and dexam ethasone together, the rise in fibrinogen was 15-20 fold, the dexam ethasone enhancing the IL-6 effect on m RN A production. Using HepG 2 cells, Rose-John and colleagues (1 9 9 0 ) showed that dexamethasone induced a time- and dose-dependent upregulation of IL-6 receptor levels which caused an earlier and increased response to IL-6 as shown by increased y-fibrinogen mRNA. They postulated that expression of the IL-6 receptor might be the rate-limiting step in the acute phase response. M azzorana and colleagues (1991) showed that the R-fibrinogen m R N A increased 5 fold in response to addition of glucocorticoids to cultured adult human hepatocytes. Huber and colleagues (1990) demonstrated a domain between

-2900 b p and -1500bp from the start of transcription which confers dexam ethasone inducibility.

T h e role of the cytokine transforming growth factor

Q>

Interleukin-1 (IL-1), another cytokine, has been shown to decrease fibrinogen production in adult human hepatocytes (Castell et al, 1989) and in H epG 2 cells (Baum ann et al, 1987) but no such effect was seen in Hep3B2 cells (Darlington et al, 1986). However, IL-1 has also been shown to induce the expression of IL-6 in a variety of cell types and possibly provides a mechanism for regulating the acute phase response in vivo (Ray et al, 1988; Zhang et al, 1988).

Both IL-1 and IL-6 have been found to stimulate a mouse pituitary tumour cell line to release adrenocorticotrophic hormone (ACTH) which in turn stimulates glucocorticoid production (Woloski et al, 1985). Dexam ethasone w as shown to inhibit the production of IL-6 by cultured monocytes (Woloski et al, 1985). These findings led Woloski and coworkers to propose that tissue injury caused monocytes to release I IL-1 and IL-6 which then stimulated A C T H and thus glucocorticoid production. These factors together elicit acute phase protein production in the liver while the glucocorticoids also inhibit IL-1 and IL-6 production thus removing the stimulation to the pituitary to release A C T H and consequently, causing a reduction in glucocorticoid levels. This provides a model for the initiation, amplification and subsequent limitation of the acute phase response.

The regulation of constitutive fibrinogen expression in liver is complex; a - and B- gene expression is mostly dependent on HNF-1 (Courtois et al, 1988) while y-gene expression is regulated by three ubiquitous nuclear proteins, S p l, major late transcription factor and CAAT-binding factor (Morgan et al, 1988). During the acute phase of inflammation, a coordinate accumulation of a, B and y m RN A is observed (Crabtree and Kant, 1982) and this effect can be mimicked by recombinant IL-6 (G eiger et al, 1988). It has been shown using pulse-chase experiments and later by transfection of the BB chain cDNA into HepG2 cells that synthesis of the BB chain is the rate limiting step in the formation of fibrinogen (Yu et a l,1983; Yu et al, 1986; Roy et al, 1990) and it is therefore reasonable to assum e that changes in

the rate of transcription of the

Q>