During acute inflammation, leukocyte recruitment is characterized by an initial infiltration of neutrophils, which are later replaced by a more sustained population of mononuclear cells. Based on both clinical and experimental evidence, we present a role for IL-6 and its soluble receptor (sIL-6R) in controlling this pattern of leukocyte recruitment during peritoneal inflammation. Liberation of sIL-6R from the initial neutrophil infiltrate acts as a regulator of CXC and CC chemokine expression, which contributes to a suppression of neutrophil recruitment and the concurrent attraction of mononuclear leukocytes. Soluble IL-6R-mediated signaling is therefore an important intermediary in the resolution of inflammation and supports transition between the early predominantly neutrophilic stage of an infection and the more sustained mononuclear cell influx.

Although fibrinogen genes are expressed constitutively in hepatocytes, their transcription can be greatly increased during inflammatory stress. Extensive studies have focused on the cytokine mediated transcriptional regulation of fibrinogen genes. It is clear that interleukin-6 (IL-6) and its family of cytokines are the major inducers of fibrinogen gene expression. Functional analyses of all three fibrinogen promoters for human and rat all demonstrate that the conserved CTGGGAA motifs within the proximal promoter of each fibrinogen gene are the IL-6 responsive elements. Exploration of the rat gamma fibrinogen gene demonstrated that the IL-6 activated transcription factor, STAT3, binds to the CTGGGAA motif and is required for the IL-6 mediated upregulation of this gene. IL-6 mediated fibrinogen production can be significantly elevated by glucocorticoid treatment. The synergistic effect of glucocorticoids and IL-6 relies on the functional interaction between STAT3 and glucocorticoid receptor. In addition to the upregulation signals for fibrinogen gene expression during inflammatory stress, other signaling also downregulates the expression of fibrinogen genes. For example, the proinflammatory cytokine IL-1 beta exerts inhibitory function on IL-6 mediated fibrinogen gene expression. Given the fact that elevated levels of fibrinogen in blood correlate with increased risk for cardiovascular disease, there is strong motivation to explore the molecular mechanisms that control fibrinogen expression, especially those signals that may downmodulate expression and thus provide novel approaches to controlling fibrinogen levels.

The suitability of existing topical fibrin glue preparations for tissue sealing or local drug delivery applications is greatly limited by their poor mechanical properties and the limited capacity of fibrinogen (Fgn) to actively bind growth factors or other therapeutic agents. Poly(ethylene glycol) (PEG) offers potential solutions to these problems by providing a mechanism for increasing the number of crosslinks between adjacent fibrin monomer molecules or for covalently crosslinking Fgn to therapeutic agents. The feasibility of this approach requires the full biological activity, or clottability, of PE glycolated Fgn. This study characterizes the clot characteristics of Fgn modified to varying degrees with monofunctional succinimidyl propionate PEG (5000 Da). The data indicate that, although thrombin clotting times are significantly altered, Fgn maintains 90% of its capacity to clot upon the addition of up to 5 PEG/Fgn. Further derivatization significantly decreases the Fgn clottability. The addition of up to 5 PEG/Fgn has little, if any, effect on the kinetics of degradation by plasmin. The results suggest that limited modification of Fgn with lysine-reactive PEG allows therapeutic enhancement of fibrin glues.

PEGylated protein conjugates submitted as therapeutic products qualify for Biologics License Applications as new chemical entities or new molecular entities. Even an improved version of an existing product must still undergo regulatory consideration as a new product. This chapter reviews historical and current regulatory guidelines specific to PEGylated proteins illustrated by examples from currently marketed products. As analytical techniques improve, regulatory authorities expect characterization methodologies of biological products undergoing regulatory review to embody current state-of-the-art capabilities. Biosimilars of existing polyethylene glycol (PEG)–protein conjugates are also subject to regulatory scrutiny, particularly with reference to the development of antidrug antibodies. This chapter highlights various regulatory considerations required for characterization, but each product will have its own unique challenges based on the combination of the specific protein and the specific PEGylation strategy.

BACKGROUND:Consumption of moderate amounts of alcohol has been reported to exert a cardioprotective effect in individuals. The exact mechanism by which this occurs has not been fully explored. Circulating levels of the clotting protein fibrinogen have been unequivocally established as an independent risk factor for vascular diseases. This study examined the effects of moderate levels of ethanol on the expression of fibrinogen in an animal model and in hepatoma cells.METHODS:Out-bred Sprague-Dawley rats were fed 5% ethanol (v/v) in their water for 4 weeks, and circulating levels of fibrinogen were measured weekly via quantitative immunoassay. H4IIE rat hepatoma cells, which constitutively produce fibrinogen, were exposed to 10 and 20 mM ethanol, and fibrinogen production was determined. The effects of ethanol on fibrinogen messenger ribonucleic acids were determined by northern gel analyses.RESULTS:Our findings demonstrate that daily consumption of moderate amounts of ethanol decreases circulating levels of fibrinogen 18% to 20%. The decrease is reversible and is not gender specific. The exposure of hepatoma cells to ethanol also diminishes fibrinogen production by 20% by decreasing the transcription of fibrinogen genes. Alcohol concentration of 10 to 20 mM did not affect hepatoma cell viability or doubling time.CONCLUSIONS:The findings indicate that one likely positive benefit of moderate ethanol consumption is to diminish the production of fibrinogen, which reduces the potential risk exerted by this protein. The site of action of ethanol is, at least in part, exerted at the level of gene transcription.

In this study, we report that an interleukin-6 (IL-6)-inducible E1A-substituting activity can be exploited for the production of infectious adenoviral particles during infection with the E1A-deleted adenovirus (Ad) Ad5dl312. The basal level of complementation can be increased by 1.5 log by induction of the HepG2 cells with recombinant human IL-6. Additionally, the IL-6-inducible E1A-substituting activity can complement E1A deletion in other cancer cell lines to render them Ad producer cells on induction with recombinant human IL-6, although the efficiency of complementation varies between cell lines. Ad5dl312 can replicate in, produce cytotoxic effect, and kill human tumor cells without addition of exogenous IL-6 in the context of tumor cells possessing an IL-6 autocrine arc, such as ovarian tumor cells. In contrast, normal human mesothelial cells isolated from normal human peritoneum lining do not support replication of Ad5dl312, even in the presence of exogenous IL-6. These results suggest that Ad5dl312 could be used as a cytotoxic agent to selectively kill tumor cells responsive to or possessing an IL-6 autocrine arc.

The soluble interleukin 6 receptor (sIL-6R) circulates at elevated levels in various diseases. This suggests that inflammatory mediators control sIL-6R release. Through examination of human neutrophils, it was found that the acute phase reactant C-reactive protein (CRP) activates a threefold increase in sIL-6R production. Maximal release occurred after 30–60 min exposure to CRP (50 μg/ml), and was mimicked by peptides corresponding to amino acid residues 174– 185 and 201–206 of native CRP. A third peptide fragment (77–82) had no effect. Differential mRNA splicing did not account for the CRP-mediated release of sIL-6R, since this isoform was not detected in conditioned media. Furthermore, stimulation of neutrophils with CRP or with peptides 174–185 or 201–206 promoted a loss of membrane-bound IL-6R, suggesting release by proteolytic shedding. The metalloprotease inhibitor TAPI had only a marginal effect on CRP-mediated sIL-6R release, suggesting that shedding occurs via a mechanism distinct from that previously reported. It well established that IL-6 stimulates the acute phase expression of CRP. Our current findings demonstrate a novel relationship between these two mediators, since CRP may affect IL-6–mediated inflammatory events by enabling formation of the sIL-6R/IL-6 complex.

Plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor, affects the processes of fibrinolysis, wound healing, and vascular remodeling. We have demonstrated that PAI-1 transcription is induced by D dimer, a plasmin proteolytic fragment of fibrin, supporting its role in negative feedback on peri-cellular proteolysis. The focus of this study was to define the mechanism of D dimer’s effects on PAI-1 transcription. D dimer increased the binding activity of the transcription factor activator protein-1 components c-fos/junD and c-fos mRNA levels in a time- and concentration-dependent manner to a greater extent than fibrinogen. Both basal and D dimer-induced PAI-1 transcriptional activity were entirely dependent on elements within the −161 to −48 bp region of the PAI-1 gene in fibroblasts. Mutations within the AP-1–like element (−59 to −52 bp) in the PAI-1 gene affected D dimer-induced transcriptional activity, c-fos/junD DNA binding, and basal and c-fos inducible PAI-1 transcriptional activity. Furthermore, expression of either wild-type or mutant c-fos proteins augmented or diminished the response of the PAI-1 promoter (−161 to +26 bp) to D dimer, respectively. D dimer-induced binding of c-fos/junD to the highly conserved and unique AP-1 like element in the PAI-1 gene provides a mechanism whereby specific fibrin fragments control fibrin persistence at sites of inflammation, fibrosis, and neoplasia.

The promoter region of the Bβ fibrinogen gene containing the polymorphic site (G−455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different −455 nucleotide substitutions of the Bβ promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position −455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at −455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bβ gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele. © 1998 by The American Society of Hematology.

Interleukin-6 (IL-6) and glucocorticoids are important mediators of inflammatory and immunological responses. Glucocorticoids are known to synergistically enhance IL-6-mediated cellular responses. We now show that IL-6 also has a synergistic effect upon glucocorticoid signaling. In particular, IL-6-activated STAT3 associates with ligand-bound glucocorticoid receptor to form a transactivating/signaling complex, which can function through either an IL-6-responsive element or a glucocorticoid-responsive element. These findings reveal a new level of interaction between these two crucial signaling cascades and indicate that activated STAT3 can also act as a transcriptional co-activator without direct association with its DNA binding motif.

Persistent fibrin deposition in tissues characterizes the early pathology of many types of injury. In an animal model of bleomycin- induced lung fibrosis, increased expression of type 1 plasminogen activator inhibitor (PAI-1) is associated with accumulation of fibrin in fibroproliferative lesions. Plasmin proteolysis of cross-linked fibrin generates fibrin degradation products (FDPs) with multiple biological activities in several cell types. We reasoned that fibrin fragments may also regulate fibroblast-mediated fibrinolysis. In this study, we describe induction of PAI-1 mRNA, protein, and activity by soluble FDPs and fibrinogen in rat lung fibroblast monolayers. FDPs are more potent than fibrinogen, inducing a concentration-dependent, maximal 3.7 (+/- 0.9)-fold increase in PAI-1 mRNA as measured by northern blotting and a 9.0 (+/- 1.3)-fold induction of PAI-1 antigen levels. Active PAI-1 is demonstrated in fibrinogen- and FDP-stimulated conditioned media. Further characterization of this response shows that PAI-1 expression is induced by the DD/D fragments, but not by immunopurified fragment E. Experiments using Actinomycin D and puromycin indicate that the induction appears to be transcriptionally regulated and is not dependent on new protein synthesis. FDP induction of PAI-1 suggests a matrix-cell feedback process in which a fibrin fragment modulates expression of an important regulator of fibrinolysis.

Bleomycin lung injury in mice leads to an acute alveolitis followed by a fibroproliferative response characterized by the accumulation of extracellular matrix. Because distinct regions of the fibrin(ogen) molecule have unique in vitro biological effects on cells, we quantified, localized, and biochemically characterized the molecular form of extravascular fibrin(ogen) in methoxyflurane anesthetized, bleomycin-injured mice. Bleomycin or saline (controls) was administered intratracheally, and lung tissue was harvested and analyzed at several times thereafter. Immunoreactive fibrin tissue content increased to a maximal 50-fold over controls in a temporal and spatial pattern paralleling that of alveolitis and maximal fibroproliferation. The generation of gamma-gamma-chain dimers and alpha-chain polymers, together with the loss of free alpha- and gamma-chains, indicates that the fibrin is predominantly covalently cross-linked. In fibroproliferative phase lungs, the fibrin fibrils are branched and colocalize with those of collagen at the electron microscopic level. These observations strongly suggest that fibrin is a significant molecular effector of the in vivo fibroproliferative response after lung injury.