Showing papers by "Richard Bucala published in 1994"

Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.

Abstract: The host response to tissue injury requires a complex interplay of diverse cellular, humoral, and connective tissue elements. Fibroblasts participate in this process by proliferating within injured sites and contributing to scar formation and the long-term remodeling of damaged tissue. Fibroblasts present in areas of tissue injury generally have been regarded to arise by recruitment from surrounding connective tissue; however this may not be the only source of these cells. Long-term culture of adherent, human, and murine leukocyte subpopulations was combined with a variety of immunofluorescence and functional analyses to identify a blood-borne cell type with fibroblast-like properties. We describe for the first time a population of circulating cells with fibroblast properties that specifically enter sites of tissue injury. This novel cell type, termed a “fibrocyte,” was characterized by its distinctive phenotype (collagen+/vimentin+/CD34+), by its rapid entry from blood into subcutaneously implanted wound chambers, and by its presence in connective tissue scars. Blood-borne fibrocytes contribute to scar formation and may play an important role both in normal wound repair and in pathological fibrotic responses.

The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor.

Abstract: For over 25 years, the cytokine known as macrophage migration inhibitory factor (MIF) has been considered to be a product of activated T lymphocytes. We recently identified the murine homolog of human MIF as a protein secreted by the pituitary in response to endotoxin administration. In the course of these studies, we also detected MIF in acute sera obtained from endotoxin-treated, T cell-deficient (nude), and hypophysectomized mice, suggesting that still more cell types produce MIF. Here, we report that cells of the monocyte/macrophage lineage are an important source of MIF in vitro and in vivo. We observed high levels of both preformed MIF protein and MIF mRNA in resting, nonstimulated cells. In the murine macrophage cell line RAW 264.7, MIF secretion was induced by as little as 10 pg/ml of lipopolysaccharide (LPS), peaked at 1 ng/ml, and was undetectable at LPS concentrations > 1 microgram/ml. A similar stimulation profile was observed in LPS-treated peritoneal macrophages; however, higher LPS concentrations were necessary to induce peak MIF production unless cells had been preincubated with interferon gamma (IFN-gamma). In RAW 264.7 macrophages, MIF secretion also was induced by tumor necrosis factor alpha (TNF-alpha) and IFN-gamma, but not by interleukins 1 beta or 6. Of note, MIF-stimulated macrophages were observed to secrete bioactive TNF-alpha. Although previously overlooked, the macrophage is both an important source and an important target of MIF in vivo. The activation of both central (pituitary) and peripheral (macrophage) sources of MIF production by inflammatory stimuli provides further evidence for the critical role of this cytokine in the systemic response to tissue invasion.

Advanced glycation end products contribute to amyloidosis in Alzheimer disease

Abstract: Alzheimer disease (AD) is characterized by deposits of an aggregated 42-amino-acid beta-amyloid peptide (beta AP) in the brain and cerebrovasculature. After a concentration-dependent lag period during in vitro incubations, soluble preparations of synthetic beta AP slowly form fibrillar aggregates that resemble natural amyloid and are measurable by sedimentation and thioflavin T-based fluorescence. Aggregation of soluble beta AP in these in vitro assays is enhanced by addition of small amounts of pre-aggregated beta-amyloid "seed" material. We also have prepared these seeds by using a naturally occurring reaction between glucose and protein amino groups resulting in the formation of advanced "glycosylation" end products (AGEs) which chemically crosslink proteins. AGE-modified beta AP-nucleation seeds further accelerated aggregation of soluble beta AP compared to non-modified "seed" material. Over time, nonenzymatic advanced glycation also results in the gradual accumulation of a set of posttranslational covalent adducts on long-lived proteins in vivo. In a standardized competitive ELISA, plaque fractions of AD brains were found to contain about 3-fold more AGE adducts per mg of protein than preparations from healthy, age-matched controls. These results suggest that the in vivo half-life of beta-amyloid is prolonged in AD, resulting in greater accumulation of AGE modifications which in turn may act to promote accumulation of additional amyloid.

Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency

Abstract: Atherosclerosis develops rapidly in patients with diabetes or renal insufficiency. Plasma lipoprotein profiles are frequently abnormal in these conditions and reflect an elevation in the level of the apoprotein B (ApoB)-containing components very low density lipoprotein (VLDL) and low density lipoprotein (LDL). High levels of circulating advanced glycation end products (AGEs) also occur in diabetes and end-stage renal disease (ESRD). These products arise from glucose-derived Amadori products and include AGE-modified peptides (AGE-peptides) which result from the catabolism of AGE-modified tissue proteins. AGE-peptides have been shown to crosslink protein amino groups and to accumulate in plasma as a consequence of renal insufficiency. To address potential mechanisms for the dyslipidemia of diabetes and ESRD, we investigated the possibility that circulating AGEs react directly with plasma lipoproteins to prevent their recognition by tissue LDL receptors. AGE-specific ELISA showed a significantly increased level of AGE-modified LDL in the plasma of diabetic or ESRD patients compared with normal controls. AGE-LDL formed readily in vitro when native LDL was incubated with either synthetic AGE-peptides or AGE-peptides isolated directly from patient plasma. LDL which had been modified by AGE-peptides in vitro to the same level of modification as that present in the plasma of diabetics with renal insufficiency exhibited markedly impaired clearance kinetics when injected into transgenic mice expressing the human LDL receptor. These data indicate that AGE modification significantly impairs LDL-receptor-mediated clearance mechanisms and may contribute to elevated LDL levels in patients with diabetes or renal insufficiency. This hypothesis was further supported by the observation that the administration of the advanced glycation inhibitor aminoguanidine to diabetic patients decreased circulating LDL levels by 28%.

Purification, bioactivity, and secondary structure analysis of mouse and human macrophage migration inhibitory factor (MIF).

Abstract: The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and the monocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF from anterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MIF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF-exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-alpha and stimulated nitric oxide production by macrophages primed with interferon-gamma. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.

Comparative analysis of DNA mutations in lacI transgenic mice with age.

Abstract: Transgenic mice have been developed recently that contain copies of the well-defined mutagenesis reporter gene, lacI, in an integrated bacteriophage-based shuttle vector. The lacI gene, which is present in all cells of the mouse, can be excised specifically from isolated genomic DNA and efficiently packaged into bacteriophage particles after the addition of packaging extracts. Mutations of the lacI gene are easily detected by the derepression of beta-galactosidase resulting in blue plaques in the presence of X-gal. Originally developed as a short-term in vivo mutagenesis assay to screen genotoxic agents, we have used this system to measure naturally occurring DNA mutations as a function of chronological age. There was a linear increase in the presence of phenotypic lacI mutants from birth to 24 months (r = 0.731, P 3 months of age demonstrated a higher percentage of mutations (12-19.4% vs. 1.2%) resulting from discriminable size changes (> 20 bp) than was observed for mice 1-2 months old. Sequence analysis of mutations resulting from a > 20 bp size change revealed them to be due to a duplication of adjacent lacI sequence. These results indicate that there is a gradual accumulation of DNA mutations with age and that the types of mutations also are influenced by the age of the animal.

Sustained and Distinctive Patterns of Gene Activation in Synovial Fibroblasts and Whole Synovial Tissue Obtained from Inflammatory Synovitis

Abstract: Fibroblastoid synovial lining cells isolated from rheumatoid and other chronic inflammatory synovial tissue exhibit distinctive and sustained alterations in serial culture not commonly found in similarly cultured cells from osteoarthritic synovium. These are demonstrable using a multi-gene dot blot assay by labelling reverse transcribed fibroblast cDNA which is hybridized to plasmids containing relevant target gene inserts. Cultured synovial fibroblastoid cells from patients with chronic inflammatory synovitis expressed significantly higher levels of stromelysin, vimentin and TIMP-1 mRNA and lower levels of c-myc compared to cells isolated from osteoarthritis synovium although with considerable variation. Early fetal synovial lining cells were similar to cells from osteoarthritis synovium but vimentin expression was higher. Marked differences in patterns of gene expression between cell lines persisted through 10 serial passages over 6-8 months. In whole synovia, the average level of mRNA for stromelysin, vimentin, IL-4, IL-6, TIMP-1, cathepsin D, gelatinase, TGF alpha, c-fms and DR beta were preferentially expressed in inflammatory tissue while c-myc expression was higher in osteoarthritis synovium. Inflammatory synovium also expressed TNF alpha, IL-1 alpha, IL-1 beta, IL-2, c-sis, tissue plasminogen activator, CSF-1, and GM-CSF. This pattern resembles, in part, that found in cultured inflammatory fibroblasts but, in addition, gene products apparently reflecting the presence of activated monocytes and lymphocytes were detected. These results provide evidence that profiles of certain gene activation in cells from patients with inflammatory synovitis differ from those with non-inflammatory disease and suggest that the fibroblastoid cells are responsible for a considerable proportion of the altered phenotypic expression pattern in whole tissue. Furthermore, this modulated pattern of gene activation appears to be an intrinsic pro-inflammatory characteristic of the fibroblastoid cells initiated in response to chronic inflammation and persists for a prolonged period in the absence of other inflammatory cells.

The emerging role of MIF in septic shock and infection.

Abstract: The protein mediator described originally as macrophage migration inhibitory factor (MIF) has been “re-discovered” recently to be both a novel pituitary hormone and a pro-inflammatory, macrophage-derived cytokine. Emerging studies indicate that MIF plays a pivotal role not only in endotoxic shock but also in the host response to a variety of acute and chronic infections.

Advanced glycosylation endproducts: Role in diabetic and non-diabetic vascular disease

Abstract: Reducing sugars such as glucose react non-enzymatically with the amino groups of macromolecules to initiate a chemical modification process known as advanced glycosylation. This pathway proceeds from reversible Schiff base adducts to a heterogenous group of irreversibly-bound, cross-linking moieties called advanced glycosylation endproducts or AGEs. Recent progress in our understanding of this process in vivo has affirmed the hypothesis that AGEs play an intimate role in the evolution of both diabetic and non-diabetic vascular disease. Utilizing newly developed AGE-specific ELISA techniques, AGEs have been identified to be present on a variety of vascular wall, lipoprotein, and lipid constituents. Protein-bound AGEs, for example, contribute to a variety of pathological effects in vivo by acting to increase vascular permeability, enhance subintimal protein and lipoprotein deposition, and exert direct toxic effects on endothelial cells. Lipid-AGEs promote oxidative modification and may contribute to atherogenesis by initiating the formation of oxidized low density lipoprotein (ox-LDL). Aminoguanidine, a recently developed pharmacological inhibitor of advanced glycosylation, is presently undergoing phase II clinical trials and may offer a specific therapeutic modality for the treatment of pathological conditions that result from excessive advanced glycosylation.