The potential targets of Kupffer cells activity during liver regeneration

This paper addresses possible causal and kinetic correlations between production of biologically active molecules by Kupffer cells and biochemical events in regenerating liver after partial hepatectomy. The expression and activity of some genes and enzymes were evaluated that are potential targets for regulation by Kupffer cells, e. g. transcriptional factors coded by the nuclear protooncogenes, c-fos and c-myc, a tissue-specific gene P450IIE1 and 2'5'oligo(A) synthetase. The results were compared with data about Kupffer cell activity during regeneration. Kupffer cells manifest their specific activities during transition of hepatocytes from quiescence to the first cell cycle and during the promotion of the last one. After termination of these functions the macrophages themselves enter into a cell cycle. These phenomena indicate a regulatory role of mesenchymal cells in triggering and promoting liver regeneration.

Introduction.An intriguing question of contempory research on liver regeneration is the particular role of sinusoidal non-parenchymal cells.They not only participate in the growth processes by increasing their number but also must be seen as participants in the metabolism of the whole regenerating organ and in the cross-talk with hepatocytes.This relation has not yet been well elucidated.Several points argue in favor of a participation of Kupffer cells (КС) that produce numerous mediators [1].With regard to the re generating process, their activation prior to partial hepatectomy (PHE) accelerates, their inhibition de lays the restoration of the parenchyma [2].Animals with genetically altered sensitivity to lipopolysaccharide (LPS) manifest weaker response to PHE [3 ].The pattern of produced mediators changes in the course of regeneration, e. g. medium conditioned by КС obtained during the first 12 h after PHE sti mulates DNA synthesis in hepatocytes [4 ] while that from nonparenchymal cells obtained on the second day after operation delays it [5 ].The expression and activity of some genes and enzymes were evaluated that are potential targets for regulation by Kupffer cells [6 ].The results were compared with data about Kupffer cell activity during regeneration.
C-Fos is a ubiquitous nuclear phosphoprotein that together with c-Jun forms stable heterodimer known as the transcriptional activator protein (AP-1).AP-1 possesses a dual function activating not only trans cription but DNA replication [7].AP-1 activity is regulated by wide spectrum of stimuli, including cytokines and growth factors.
C-Myc is a nuclear phosphoprotein that together with c-Max forms a transcriptional factor.The func tional activity of c-Myc is regulated at transcriptional, posttranscriptional and posttranslational levels.c-Myc is critical for proliferation/differentiation balance and cell cycle progression especially during Gl and S-to-G2/M phase [8 ].
P450IIE1 is a microsomal enzyme involved in metabolism of foreign compounds.Cytokines IL-1, IL-6, TNF-a and IFNa/p downregulate its expression [9].2'5'oligo(A) synthetase is a limiting enzyme of 2'5' oligo(A)synthetase/RNAse L system negatively regulating the amount of RNA at the pretranslational level.The c-myc RNA is a potential target of the system.Cytokines IFNa//? and TNF-a positively regulate enzymes expression [6,10].
The most reliable time marker of regenerating process is the DNA synthesis in the liver cells.Hepatocytes, КС and sinusoidal endothelial cells enter their first cell cycles in the given order [11].The S phase of hepatocytes (ca.12-25 h after PHE) is preceeded by prereplicative period conveniently subdivided into immediate-early response (ca.0.5 h after PHE), delayed reaction with metabolic reorientation providing competence to proliferate (ca.0.5-3 h after PHE) and a Gl-like period (ca.3-12 h after PHE) [12].The G2 and M phases in hepatocytes have standard periods of 4.5 h and 1 h, respectively.
Materials and Methods.Pretreatment of the ani mals.Male Wis tar rats (150-200 g) were used throughout.Partial (2/3) hepatectomy and sham operation were performed by standard procedure.Livers were perfused in situ at the time indicated with cold physiological saline and were processed either immediately or frozen and stored at -70°C before usage.
Detection of relative amount of specific RNA.Total RNA from nuclear and cytoplasmic (S10) liver fractions was isolated by a slightly modified guanidinium isothiocyanate method.Poly (A)"cytoplasmic RNA was separated on oligo(dT) cellulose, the rela tive amount of specific RNAs was registered by dot-, blot hybridizations in the presence of formamide.The probes were radioactively labeled with [ 32 P ]-dCTP in nick-translation or randomly primed reactions.The intensity of hybridization signals was registered in autographs with a laser scanner.The following probes were used: the PstI fragment (1100 bp) of the v-fos gene, the Xbal-SacI fragment of murine c-myc cDNA and the PstI fragment (300 bp) of murine P450IIE1 cDNA.
Detection of 2'5'oligo(A)synthetase activity.En zymatic activity of the nuclear (SI2) and cytoplasmic (SI5) fractions was detected using the specific bin ding activity of polyl-polyC paper and catalytic acti vity of bound enzyme.2'5'oligo (A) adenylates were synthesized in the presence of [ M C]-ATP and were separated by chromatography on PEI-cellulose.Their radioactivity was measured [13].
Results.The relative amounts of c-fos, c-myc and P450IIE1 specific RNAs.Nuclear and poly (A) Cy toplasmic RNAs from intact, regenerating and sham operated liver produced hybridization signals with each of these probes.The nuclear RNAs existed as precursor molecules of greater molecular weight (data not shown).The time course of changes is specific for each RNA.The kinetics of nuclear and poly (A) + cytoplasmic P450IIE1 RNA are similar -the upregulation during the first three hours after PHE is followed by down-regulation during 6-24 h interval and slight increase later on (fig. 1, 4).This coin cidence suggests the prevalence of transcriptional over posttranscriptional control of gene expression.Downregu lation during late Gl-S phase of hepatocytes correlates with resistance of regenerating liver to he patotoxins [14].Nuclear c-fos and c-myc specific RNAs are up-regulated with the maxima at 3 h and ca.18 h after PHE (fig.2).Their counterparts in poly (A) + cytoplasmic RNA do not follow the same time course (fig.3), especially during the prere- plicative period; this raises the questions about an involvement of posttranscriptional events in the regu lation of these genes.Both poly(A) + RNAs are downregulated after their immediate increase after PHE, later on, the relative amount of c-myc RNA is elevated during the Gl-to-Gl/S and S-to-G2/M tran sitions (fig.3), that of c-fos RNA during the S phase of hepatocytes (fig.4).Immediate-early transient expression of c-fos and c-myc transcripts is well documented for different cells in particular for mitogen-stimulated hepatocytes [15].The expression of these genes during the later periods of regeneration remains to be clarified.The increase of c-fos trans cripts during the S phase of hepatocytes implicates c-Fos participation in DNA synthesis or related 40 processes and correlates with activating role of AP-1 in DNA replication [7 ].The time-course of c-myc transcripts in regenerating liver resembles the similar changes of c-Myc -c-Max transcriptional activity and phosphorylation pattern of c-Myc in proliferating cells [8].
2'5'oligo(A)synthetase activity after PHE.Enzy me activity was detected in nuclear and cytoplasmic fractions, the nuclear specific activity being higher by one order (fig.5).Taking into account the distri bution of the cellular protein between both fractions, their input to the total cellular enzymatic activity is nearly equal.PHE induced a "temporal decrease in nuclear activity with minimum at 3 h (compare the up-regulation of c-fos, c-myc and P450IIE1 nuclear transcripts at this time (fig.2), and an increase in the cytoplasmic activity with a maximum at 0.5 h after PHE (compare the simultaneous decline of c-myc transcripts in cytoplasm) (fig.3).It should be noted that the reorganization of ribosomal machinery occurs also at 0.5 h after operation.The ribosomes tran siently lose attachment to the rough endoplasmatic reticulum.The restoration of the typical structure of the latter begins 1 h after PHE [12].
Discussion.The biological activities of mediators, their availability in each period of the process and the corresponding biochemical events in regenerating liver have been analyzed.The available evidences about КС activity during liver transition from quiescence to proliferation and during their first cell cycle are summarized in table.The production of thromboxane A 2 (T*A 2 ) was up-regulated during the immediateearly response (see table); this eicosanoid is produced in the liver mainly by КС from the endogenous pool of bound arachidonic acid in response to КС sti mulation by phagocytosis or intracellular Ca" ele vation 11 J.The ability of thromboxane to induce glycogenolysis, platelet activation and sinusoids cons truction [ 1 ] is in line with the processes occuring at this time in the liver [16].The nature of the earliest up-regulation of nuclear protooncogenes expression is not yet clear.
During the delayed-early response, the action of potent cytokines, e. g.TNF-a and IL-1 [17][18][19][20], and the increased responsiveness to them due to elevated expression of TNF receptors [17] may define the whole set of observed events, e. g. up-regulation of c-fos-and c-myc-and down-regulation of P450IIE1 expression, as demonstrated here, NO production in hepatocytes as shown previously [21,22], proin flammatory and hepatocyte-specific promitogenic re actions, and autostimulation of PGE 2 synthesis [23,24 ].Prostaglandin E 2 is instrumental in the re gulation of cytokine production [23 ], the increase of intracellulr cAMP [23 ], in relaxation of sinusoids and consequently enhanced blood flow in the liver [25].IFNa//? and TNF-a are potential regulators of 2',5'oligo(A)synthetase activity [10], but differential regulation of enzyme in nucleus and in cytoplasm points to the complexity of this regulation.

Kupffer cells activity in regenerating liver
During the Gl-like period, the set of acting cytokines enlarged by the two complete mitogens, transforming growth factor a and hepatocyte growth factor (HGF); counteracting is TGF-£, an inhibitor of DNA synthesis [15].They originated in regenerating liver from hepatocytes, endothelial cells and hepatic stellate cells, respectively [15].Whether and to what extent КС are implicated in this synthesis is not yet clear.Their role as stimulators of HGF expression via TNF-a, however, is established [25].The up-regu lation of c-fos and c-myc expression, the sustained down-regulation of P450IIE1 gene expression and the enhanced production of NO by hepatocytes are con sistent with an involvement of КС.
During the S phase of hepatocytes КС activity slowly declines.The progressively increasing pro duction of PGE 2 seems to inhibit TNF-a and IL-1 syntheses by КС [ 1 ].The liver macrophages obtained at this time synthesize in vitro less cytokines than the cells from sham-operated animals while blockade of cyclooxygenase pathway greatly activates them [24 ].The involvement of КС in stimulation of IL-6 synthesis at this or previous stage is also likely [19].IL-6 is the most potent regulator of the acute phase response that temporally coincides with the S phase [27].Despite the fact that stimulated КС produce IL-6 [1], their involvement in synthesis occuring in regenerating liver has not yet been proved.
Two other sets of evidences complete the scheme of regenerating process.Previously, we have shown that NO production coincides with the entrance of hepatocytes into the cell cycle [21 ].While NO production in hepatocytes is under cytokines control [22], NO synthesis in activated КС is under PGE 2 control [ 1 ].It seems likely that the high level of PGE 2 triggers NO production in КС manifesting their entrance into the cell cycle.Endogeneous NO is known as inhibitor of cyclooxygenase pathway and IL-6 producion in КС [28].If it is also the case in the regenerating liver, the paracrine activity of КС is suppressed giving the way for the cell cycle-connected processes.
Thus, the cell-specific actions of Kupffer ceils provide the regulatory network in the regenerating liver.The synergism between cell-specific functions of Kupffer cell and non-parenchymal cells in general and proliferative activity of hepatocytes is a key mechanism of liver regeneration.This statement is compatible with the general idea about the regulatory role of mesenchymal cells in the functioning of epithelial cells.