The growing complexity of cytokine and hormonal regulation of the liver acute phase (AP) response provides a serious challenge to this area of research. The name "interleukin", implying a communication signal between leukocytes, is clearly misleading and is being gradually replaced by "cytokine", which emphasizes cell origin and various cellular targets. The situation is complex, since free radicals can directly affect cytokine-producing cells or modifying proteins in the intercellular fluids. The promoter region of the human IL-6 gene contains several regulatory elements, and many factors can induce expression of this model cytokine. Cellular receptors for cytokines show a high degree of specificity, but they share common structural features and belong to hematopoietin receptor superfamilies. Proteins modified by free radicals or leukocyte or lysosomal proteinases will then be taken up by "scavenger-type" or specific receptors present on macrophages, fibroblasts, or endothelial cells, and by an unknown mechanism switch on the synthesis of AP-initiating cytokines.

The proteasome is a protein complex responsible for the degradation of polyubiquitin-tagged proteins. Besides the removal of target proteins, the proteasome also participates in the regulation of gene transcription in both proteolytic and non-proteolytic fashion. In this study the effect of proteasome inhibition on the basal expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1) was examined. Treatment of HepG2 or HeLa cells with proteasome inhibitor MG-132 resulted in a significant increase of MCPIP1 expression, both at mRNA and protein level. Interestingly, MG-132 did not alter MCPIP1 stability. Instead, the observed protein increase was blocked by actinomycin D, suggesting the involvement of denovo mRNA synthesis in the increase of MCPIP1 protein following MG-132 treatment. Using several inhibitors we determined the participation of extracellular-signal-regulated kinase 1/2 and p38 kinases in MCPIP1 upregulation by MG-132. Our findings show for the first time the impact of proteasome inhibition on MCPIP1 protein expression by modulation of the activity of intracellular signaling pathways. Overexpression of MCPIP1-myc protein decreased the viability of HeLa cells but not HepG2 cells, which correlates with the increased susceptibility of HeLa cells to MG-132 toxicity. Notably, both MG-132 treatment and MCPIP1-myc overexpression led to the activation of apoptosis, as revealed by the induction of caspases 3/7 in both types of cell lines. This suggests the involvement of MCPIP1 upregulation in toxic properties of proteasome inhibition, which is an acknowledged approach to the treatment of several cancer types.

The generalized inflammatory response leads to activation of hundreds of genes transcribed in an established sequence in specialized cells. Transcriptome analysis of human monocyte-derived cells stimulated with IL-1beta or with monocyte chemotactic protein-1 (MCP-1) has led to the identification of a new inflammation-related gene ZC3H12A encoding a chain of 599 amino acids corresponding to a 66-kDa protein. The protein, given a provisional name of MCPIP1 (monocyte chemotactic protein-induced protein-1), is expressed in several human and murine tissues such as bone marrow, spleen, heart and placenta. In in vivo studies, mice with inactivated MCPIP1-encoding gene showed growth retardation, lymphadenopathy, splenomegaly and enhanced inflammatory symptoms. Principal molecular features of MCPIP1 include a single zinc finger motif, an RNase-like PIN domain and ubiquitin-binding domain. Reports from independent laboratories suggest that MCPIP1 may function also as a deubiquitinase. Although MCPIP1 is regarded by some authors as a new transcription factor or cell differentiation factor modulating angiogenesis or adipogenesis, its principal function appears to be downregulation of inflammatory responses through at least two independent mechanisms: increased degradation of cytokine mRNAs and inhibition of LPS- and IL-1-induced NF-kappaB signaling pathway. The interference with NF-kappaB activation is highly complex and includes TRAF6 and TANK interaction with the ubiquitin-associated (UBA) domain of MCPIP1. Purified MCPIP1 protein was reported to degrade specific mRNA and cleave K48- and K63-linked polyubiquitin chains. Although some structural features and the mechanism of action of MCPIP1 are not fully explained yet, its importance in the regulation of inflammatory reactions has been firmly established.

Inflammation is usually defined as a complex response of the animal organism to tissue injury or to invasion of foreign pathogens. Although inflammation often starts as a local reaction it can easily develop into a generalized systemic response involving the whole organism. Participation of inflammatory reactions in the pathogenesis of many diseases is today a generally accepted medical paradigm. These ideas have been thoroughly discussed in numerous excellent monographs and specialized recent publications (Nathan, 2002; Dinarello, 2008, 2010; Medzhitov, 2010) whereas our presentation is just a brief account focused on regulatory mechanisms involved in the initiation, development and termination of the “generalized inflammatory response” (GIR) and its relations to the induced synthesis of certain plasma and tissue proteins. The results obtained by techniques of modern genomics indicate that these three phases of response to injury are tightly controlled and are characterized by distinct profiles of hundreds of activated genes. The fine tuning of gene expression during initiation, development and resolution of inflammation occurs at many levels starting from transcription and followed by changes in mRNA decay, receptors cross-talk, intracellular signaling cascades and posttranslational protein modifications. Disturbances in the regulatory network lead to either acute pathological states such as septic shock, or to various chronic autoimmune and auto-inflammatory diseases. Discovery of C-reactive protein (CRP) in the blood of patients with febrile diseases some 80 years ago (Tillett & Francis, 1930) was seminal for the development of the idea of “acute phase reaction” (APR) and the concept of “acute phase proteins” (APPs) reflected as profound rearrangement of plasma protein profile accompanying systemic inflammatory reaction (Kushner, 1982). The so called “positive AP-proteins” increase their plasma concentration in a broad range – from barely 25 per cent above the control up to several hundred fold at the expense of “negative APPs” which go down in comparison to control. Other metabolic changes during this response include fever, leukocytosis, negative nitrogen balance, altered levels of some ions and hormones, activation of clotting and complement pathways, as well as certain less defined phenomena (for references see (Gordon and Koj, 1985)). The principal features of APR resemble the unspecific innate immunological reaction

Cytokine receptors exist in membrane bound and soluble form. While most soluble receptors are antagonists, some soluble receptors are agonists like soluble receptors of the gp130 cytokine family. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. This process has been named trans-signaling [1]. We have shown that soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses. The recombinant soluble gp130 protein is a molecular tool to discriminate between gp130 responses via membrane bound and soluble IL-6R responses. We have constructed a fusion of soluble gp130 and the Fc portion of human IgG1. This sgp130Fc protein proved to be efficient in blocking responses via the IL-6/soluble IL-6R complex without affecting IL-6 responses, which are mediated via the membrane bound IL-6R [1]. The soluble IL-6R is mostly generated by proteolysis of the IL-6R transmembrane protein. Shedding of the IL-6R is mediated by the metalloprotease ADAM17, which is also responsible for the cleavage of TNFα and ligands of the EGF-R. We generated hypomorphic ADAM17 mice, which have undetectable ADAM17 protein levels in all tissues but which are still viable. Using these mice in different inflammation models we could show that activation of ADAM17 has different effects on the activation of the immune response as well as on induction of regenerative responses [2, 3]. Therefore, ADAM17 is a molecular switch of inflammatory and regenerative responses of the body to stress [3]. Using the sgp130Fc protein or sgp130Fc transgenic mice we further demonstrate that in several chronic inflammatory diseases and cancers including inflammatory bowel disease, peritonitis, rheumatoid arthritis, colon cancer, ovarian cancer and pancreatic cancer, IL-6 trans-signaling via the soluble IL-6R is a crucial step in the development and the progression of the disease. Therefore, sgp130Fc is a promising novel therapeutic agent for the treatment of chronic inflammatory diseases and cancer [1, 4-6]. References 1. Jones SA, Scheller J, Rose-John S (2011) J Clin Invest, in press. 2. Chalaris A, Adam N, Sina C, Rosenstiel P, Lehmann J, Schirmacher P, Hartmann D, Cichy J, Gavrilova O, Schreiber S, Jostock T, Matthews V, Häsler R, Becker C, Neurath MF, Reiß K, Saftig P, Scheller J, Rose-John S (2010) J Exp Med 207: 1617-1624. 3. Scheller J, Chalaris A, Garbers C, Rose-John S (2011) Trends Immunol, in press. 4. Lesina M, Kurkowski MU, Ludes K, Rose-John S, Treiber M, Klöppel G, Yoshimura A, Reindl W, Sipos B, Akira S, Schmid RM, Algül H (2011) Cancer Cell 19: 456-469. 5. Schiechl G, Bauer B, Fuss IJ, Lang SA. Moser C, Rose-John S, Neurath MF, Geissler E, Schlitt HJ, Strober W, Fichtner-Feigl S (2011) J Clin Invest 121: 1692-1708. 6. Lo C-W, Chen MW, Hsiao M, Wang S, Chen C-A, Hsiao S-M, Chang J-S, Lai T-C, Rose-John S, Kuo M-L, Wei L-H (2011) Cancer Res 71: 424-34. L1.2

The acute phase reaction is currently regarded as systemic inflammatory response and a component of innate unspecific immunological reaction. This complex response is regulated mainly by cytokines - pleiotropic mediators of metabolic and immunological reactions. Among cytokines representing various protein families and participating in inflammation one can distinguish pro-inflammatory and anti-inflammatory cytokines as well as a large interleukin-6 family occupying intermediate position. In the initiation of acute phase reaction participate various components such as Toll-like receptors recognizing foreign pathogens, inflammasomes activating interleukin-1, and protein kinases from the MAP family that finally activate transcription factor NF-kappaB involved in cytokine synthesis. Secreted cytokines are recognized by receptors of target cells and switch on expression of many genes, including those coding for acute phase proteins. Regulation of acute phase response is precise and includes not only transcription and translation but also stability of mRNA and its degradation, as well as inhibitory activity of microRNA. Disturbances in the course of acute phase reaction are responsible for auto-immunological and autoimmune diseases and various pathological states such as acute septic shock or chronic arthritis. Some acute phase proteins, and especially C-reactive protein, have important implications for clinical diagnostics and preventive medicine.

Background The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin – a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners. Results By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18–24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway. Conclusion Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.

A novel gene ZC3H12A, encoding MCP‐1‐induced protein 1 (MCPIP), was recently identified in human peripheral blood monocytes treated with monocyte chemotactic protein 1 (MCP‐1) and in human monocyte‐derived macrophages stimulated with interleukin (IL)‐1β. These experiments revealed that the gene undergoes rapid and potent transcription induction upon stimulation with proinflammatory molecules, such as MCP‐1, IL‐1β, tumour necrosis factor α and lipopolysaccharide. Here we show that the induction of ZC3H12A by IL‐1β is predominantly NF‐κB‐dependent because inhibition of this signalling pathway results in the impairment of ZC3H12A transcription activation. Our results indicate the presence of an IL‐1β‐responding region within the second intron of the ZC3H12A gene, which contains four functional NF‐κB‐binding sites. Therefore, we propose that this transcription enhancer transduces a ZC3H12A transcription‐inducing signal after IL‐1β stimulation. Recent reports suggest that MCPIP acts as a negative regulator of inflammatory processes because it is engaged in the degradation of transcripts coding for certain proinflammatory cytokines. Our observations provide evidence for a novel negative feedback loop in the activation of NF‐κB and point to potential significance of MCPIP in the treatment of various pathological states, such as diabetes or cancer that involve disturbances in the functioning of the NF‐κB system.

In human monocyte‐derived macrophages, the MCPIP gene (monocyte chemoattractant protein‐induced protein) is strongly activated by interleukin‐1β (IL‐1β). Using bioinformatics, a PIN domain was identified, spanning amino acids 130‐280; such domains are known to possess structural features of RNases. Recently, RNase properties of MCPIP were confirmed on transcripts coding for interleukins IL‐6 and IL‐12p40. Here we present evidence that siRNA‐mediated inhibition of the MCPIP gene expression increases the level of the IL‐1β transcript in cells stimulated with LPS, whereas overexpression of MCPIP exerts opposite effects. Cells with an increased level of wild‐type MCPIP showed lower levels of IL‐1β mRNA. However, this was not observed when mutant forms of MCPIP, either entirely lacking the PIN domain or with point mutations in this domain, were used. The results of experiments with actinomycin D indicate that lower levels of IL‐1β mRNA are due to shortening of the IL‐1β transcript half‐life, and are not related to the presence of AU‐rich elements in the 3′ UTR. The interaction of the MCPIP with transcripts of both IL‐1β and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes.

The transcriptome profile of human monocyte-derived macrophages stimulated in vitro by low doses of IL-1 or IL-6 was analyzed by microarrays (Affymetrix, HG-U133A) in 5 independent experiments. Out of 4886 probe sets consistently detected in all 5 array replicates we found approximately 300 genes (FDR<5%) modulated by IL-1 and/or IL-6, among which 34 may be regarded as novel cytokine-responsive macrophage genes of various function. Detailed analysis indicates that cytokine-responsive genes include 125 transcripts significantly up-regulated by IL-1 and only 39 transcripts up-regulated by IL-6, whereas the number of down-regulated transcripts is lower and almost equal for both cytokines. These data indicate that, in comparison to liver cells, IL-1 is more potent than IL-6 in modulating gene expression of human macrophages. Hierarchical clustering analysis of these transcripts yielded 7 separate gene clusters. The most abundant group contains genes strongly activated by IL-1 alone and coding for chemokines, cytokines and their receptors, the components of intracellular signaling as well as transcription factors from NF-kB family. In order to validate the results obtained by microarray analysis the expression of 5 genes from various clusters was determined by quantitative RT-PCR. Moreover, the putative promoter regions of all cytokine-responsive genes were subjected to the in silico identification of transcription factor binding sites (TFBS). We found that TFBS corresponding to RelA/NF-kB is the most strongly over-represented group and we demonstrated involvement of NF-kB in the expression of selected genes.

The discovery of C-reactive protein (CRP) almost 80 years ago in the blood of patients with various infectious and inflammatory diseases initiated studies on the mysterious biological phenomenon called “the acute phase response” (APR). The complex metabolic alterations accompanying APR include changes in the plasma concentration of the majority of liver-produced proteins – acute phase proteins (APPs). The search for mediators released from the site of injury and able to stimulate hepatocytes led to the discovery of “Leukocytic Endogenous Mediator” (or ”Endogenous Pyrogen”), initially identified with interleukin-1. Only in 1987 was the main factor able to induce the majority of symptoms of APR discovered and named interleukin-6 (IL-6). The current paradigm assumes that APR is elicited by numerous proinflammatory cytokines, the principal role being played by the IL-6-family. The last decade has brought about significant progress in understanding the initiation of the acute phase response (recognition of pathogens by Toll-like receptors), interaction of cytokines and their receptors (origin and importance of soluble cytokine receptors, construction of hypercytokines by protein fusion), the mechanism of multi-step signal transduction from the plasma membrane to nuclear transcription factors (including the role of MAP kinases), as well as elucidation of the role of receptor cross-talk in cytokine networks in health and disease. Genomic techniques indicate that hundreds of genes participate in the development of APR. The data presented here emphasize the growing importance of APR for clinical medicine and confirm the close relationship between acute phase response and innate immunity. Keywords: Acute phase proteins, pro-inflammatory cytokines, cytokine pleiotropy and redundancy, soluble receptors and engineered hypercytokines, Toll-like receptors and pathogen recognition, cytokine networks and receptor cross-talk

Among both laymen and active scientists an opinion prevails that the development of science occurs predominantly by the accumulation of knowledge and a gradual improvement in the picture of nature due to the application of new methods and techniques. The breakthroughs, such as the Big Bang theory in cosmology or deciphering of the Biological Code in the life sciences, are treated as rare exceptions. However, a closer scrutiny of almost any scientific discipline points to a noncontinuous process of development and the appearance of paradigms which are constantly subjected to verification as postulated by Kuhn (1970) in the original theory of „scentific revolutions”.

Interleukin-1 and interleukin-6 are principal cytokines involved in regulation of expression of acute-phase proteins. In the joint action of both cytokines IL-1 can suppress or enhance the IL-6-dependent induction of gene expression. Here, we report changes in the transcriptome profile of HepG2 cells exposed to IL-6 alone, or IL-1 and IL-6. Cytokine-responsive genes were identified by differential display analysis. Validation of observed changes in the transcript level was carried out using the slot blot method. Out of 88 cDNA species modulated by IL-6, only 38 represent different known genes whereas 18 clones match genomic clones in NCBI data with hypothetical cDNA sequences (the remaining 32 clones showed no homology with the database or represented several clones of the same gene). In the experiments with HepG2 cells prestimulated for 3 h with IL-1 and then stimulated with IL-6, 43 cDNA fragments were amplified. Twenty-three of them represent known genes while 10 clones have inserts matching hypothetical cDNA sequences in NCBI data. The identified transcripts modulated by IL-6 or both cytokines in HepG2 cells code for intracellular proteins of various function. The largest groups represent genes engaged in metabolism, protein synthesis and signaling pathways. Among all genes identified as differentially regulated under stimulation by IL-6, or IL-1/IL-6, six were detected in both types of stimulation. None of the typical genes coding for plasma acute phase proteins was identified in our experiments. This indicates that differential display cannot be used to characterize the profile of a given transcriptome. On the other hand, it is a useful technique for detection of new genes responding to IL-6 alone or IL-6 in combination with IL-1.

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10 ng/ml) and IL-6 (10 ng/ml). After 24 h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera.

Linear models based on proportionality between variables have been commonly applied in biology and medicine but in many cases they do not describe correctly the complex relationships of living organisms and now are being replaced by nonlinear theories of deterministic chaos. Recent advances in molecular biology and genome sequencing may lead to a simplistic view that all life processes in a cell, or in the whole organism, are strictly and in a linear fashion controlled by genes. In reality, the existing phenotype arises from a complex interaction of the genome and various environmental factors. Regulation of gene expression in the animal organism occurs at the level of epigenetic DNA modification, RNA transcription, mRNA translation, and many additional alterations of nascent proteins. The process of transcription is highly complicated and includes hundreds of transcription factors, enhancers and silencers, as well as various species of low molecular mass RNAs. In addition, alternative splicing or mRNA editing can generate a family of polypeptides from a single gene. Rearrangement of coding DNA sequences during somatic recombination is the source of great variability in the structure of immunoglobulins and some other proteins. The process of rearrangement of immunoglobulin genes, or such phenomena as parental imprinting of some genes, appear to occur in a random fashion. Therefore, it seems that the mechanism of genetic information flow from DNA to mature proteins does not fit the category of linear relationship based on simple reductionism or hard determinism but would be probably better described by nonlinear models, such as deterministic chaos.