Biomedical subjects
Publications and source records attributed to R Bucala.
The protein mediator described originally as macrophage migration inhibitory factor has been "rediscovered" to be both a novel pituitary hormone and a pro-inflammatory, macrophage-derived cytokine. MIF plays a pivotal role in the host response to endotoxic shock and appears to serve as a pituitary "stress" hormone that regulates systemic inflammatory responses.
The cytokine macrophage migration inhibitory factor (MIF) is a major protein constituent of the anterior pituitary gland released into the bloodstream during endotoxaemia. For many years, MIF had been thought to be a T cell product associated with delayed-type hypersensitivity reactions. The identification of MIF as a pituitary 'stress' hormone provides an important link in the regulation of systemic inflammatory responses by the central nervous system.
Synovial pannus represents a hypertrophic and locally invasive connective tissue response to chronic inflammation that accounts in large part for the periarticular destruction of rheumatoid arthritis. Synovial fibroblasts cultured from rheumatoid synovia have been found to display an increased rate of proliferation and the constitutive expression of collagenases, growth factors, and inflammatory cytokines. The existence in rheumatoid synovium of both a pro-inflammatory state and growth dysregulation led us to investigate the expression by synovial fibroblasts of the closely homologous cytokines GRO alpha (gro/MGSA), GRO beta (MIP-2 alpha), and GRO gamma (MIP-2 beta). These cytokines are released by a variety of cell types and display overlapping growth regulatory and pro-inflammatory activities. In contrast to expectations, the majority of synovial fibroblast cell lines derived from osteoarthritic or non-inflammatory synovia showed a relative increase in the constitutive expression of GRO alpha and GRO beta when compared to synovial fibroblasts obtained from rheumatoid synovia. Considered together with evidence that GRO alpha is a growth regulator that modulates the expression of metalloproteinase activity, these findings provide evidence for a differential pathway of cytokine activation that may downregulate the proliferative and erosive response to chronic arthritis.
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 < 0.001), such that 24-month-old mice have accumulated approximately fourfold more mutants than newborn pups. Molecular analysis of these spontaneously arising DNA mutations showed them to result predominantly from base substitutions (80.6-98.5%) that were equally distributed between transitions and transversions. However, lacI mutations in animals > 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.
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.
Recent studies have led to the re-discovery of the protein originally described as macrophage-migration inhibitory factor (MIF) to be both a pituitary hormone and a pro-inflammatory, macrophage cytokine. MIF is a pivotal mediator in endotoxic shock and may serve as a pituitary "stress" hormone that regulates systemic inflammatory responses.
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BACKGROUND: 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 longterm 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Blood-borne fibrocytes contribute to scar formation and may play an important role both in normal wound repair and in pathological fibrotic responses.
Cytokines are critical in the often fatal cascade of events that cause septic shock. One regulatory system that is likely to be important in controlling inflammatory responses is the neuroendocrine axis. The pituitary, for example, is ideally situated to integrate central and peripheral stimuli, and initiates the increase in systemic glucocorticoids that accompanies host stress responses. To assess further the contribution of the pituitary to systemic inflammatory processes, we examined the secretory profile of cultured pituitary cells and whole pituitaries in vivo after stimulation with bacterial lipopolysaccharide (LPS). Here we identify macrophage migration inhibitory factor (MIF) as a major secreted protein release by anterior pituitary cells in response to LPS stimulation. Serum analysis of control, hypophysectomized and T-cell-deficient (nude) mice suggests that pituitary-derived MIF contributes to circulating MIF present in the post-acute phase of endotoxaemia. Recombinant murine MIF greatly enhances lethality when co-injected with LPS and anti-MIF antibody confers full protection against lethal endotoxaemia. We conclude that MIF plays a central role in the toxic response to endotoxaemia and possibly septic shock.
To address potential mechanisms for oxidative modification of lipids in vivo, we investigated the possibility that phospholipids react directly with glucose to form advanced glycosylation end products (AGEs) that then initiate lipid oxidation. Phospholipid-linked AGEs formed readily in vitro, mimicking the absorbance, fluorescence, and immunochemical properties of AGEs that result from advanced glycosylation of proteins. Oxidation of unsaturated fatty acid residues, as assessed by reactive aldehyde formation, occurred at a rate that paralleled the rate of lipid advanced glycosylation. Aminoguanidine, an agent that prevents protein advanced glycosylation, inhibited both lipid advanced glycosylation and oxidative modification. Incubation of low density lipoprotein (LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components. Oxidized LDL formed concomitantly with AGE-modified LDL. Of significance, AGE ELISA analysis of LDL specimens isolated from diabetic individuals revealed increased levels of both apoprotein- and lipid-linked AGEs when compared to specimens obtained from normal, nondiabetic controls. Circulating levels of oxidized LDL were elevated in diabetic patients and correlated significantly with lipid AGE levels. These data support the concept that AGE oxidation plays an important and perhaps primary role in initiating lipid oxidation in vivo.
Advanced glycosylation endproducts react with DNA and cause mutations and DNA transposition in bacteria. To investigate the mutagenic effect of advanced glycosylation in mammalian cells, plasmid DNA containing the lacI mutagenesis marker was modified by advanced glycosylation endproducts in vitro, transfected into murine lymphoid cells, recovered, and analyzed for mutations, plasmid size changes, and the presence of shared insertion sequences. An 853-bp host-derived DNA sequence, designated INS-1, was identified as an insertion element common to plasmids recovered from multiple independent transfections. Modification of DNA by advanced glycosylation increased by 60-fold the apparent frequency of INS-1 transposition: from 0.025% to 1.5%. The INS-1 element contains a 180-bp region that is homologous to the Alu repetitive sequence family. INS-1 was also observed to be present within larger insertional mutations and, in two cases, an apparently truncated version of INS-1 that lacks the Alu region was identified. These results demonstrate the experimental induction of DNA transposition involving mammalian chromosomal elements and suggest that advanced glycosylation may play a role in the formation of Alu-containing insertions that have been found to disrupt human genes.
DNA amplification has identified P. carinii DNA in diverse biologic specimens, including the serum of patients with P. carinii pneumonia. To further examine the relationship between P. carinii DNA in serum and P. carinii infection, the corticosteroid-treated rat model of pneumocystosis was studied. By 4 weeks of immunosuppression, P. carinii DNA was detected in rat lungs and by 6 weeks, in their serum. P. carinii DNA persisted in lung tissue as long as 5 months after the withdrawal of steroids. Serum DNA disappeared 2 weeks after steroids were withdrawn. Nonimmunocompromised, sentinel rats housed near immunocompromised, P. carinii-infected rats also were studied. Within 6 weeks, P. carinii DNA became detectable in lung and by 8 weeks, in serum. P. carinii DNA disappeared rapidly from lungs and sera after sentinel rats were isolated away from corticosteroid-treated rats. These findings support the contagious transmission of P. carinii and suggest facile development of P. carinii carrier states.
Advanced glycosylation end products (AGEs) accumulate on long-lived extracellular matrix proteins and have been implicated in the micro- and macrovascular complications of diabetes mellitus. Within the arterial wall, AGE-modified proteins increase vascular permeability, inactivate nitric oxide activity, and induce the release of growth-promoting cytokines. Recently developed anti-AGE antibodies were used in an immunohistochemical analysis of coronary arteries obtained from type II diabetic and nondiabetic patients. High levels of AGE reactivity were observed within the atherosclerotic plaque present in vessels from selected patients with diabetes. Considered together with the pathological effects of AGEs on vascular wall homeostasis, these data support the role of advanced glycosylation in the rapidly progressive atherosclerosis associated with diabetes mellitus.
Advanced glycosylation end products (AGEs) have been implicated in many of the complications of diabetes and normal aging. Markedly elevated vascular tissue and circulating AGEs were linked recently to the accelerated vasculopathy of end-stage diabetic renal disease. To determine the pathogenic role of AGEs in vivo, AGE-modified albumin was administered to healthy nondiabetic rats and rabbits alone or in combination with the AGE-crosslink inhibitor aminoguanidine. Within 2-4 weeks of AGE treatment, the AGE content of aortic tissue samples rose to six times the amount found in controls (P < 0.001). Cotreatment with aminoguanidine limited tissue AGE accumulation to levels two times that of control. AGE administration was associated with a significant increase in vascular permeability, as assessed by 125I label tracer methods. This alteration was absent in animals that received aminoguanidine in addition to AGE. Significant mononuclear cell migratory activity was observed in subendothelial and periarteriolar spaces in various tissues from AGE-treated rats compared to normal cellularity noted in tissues from animals treated with aminoguanidine. Blood pressure studies of AGE-treated rats and rabbits revealed markedly defective vasodilatory responses to acetylcholine and nitroglycerin compared to controls (P < 0.001), consistent with marked NO. inactivation; aminoguanidine treatment significantly prevented this defect. These in vivo data demonstrate directly that AGEs, independent of metabolic or genetic factors, can induce complex vascular alterations resembling those seen in diabetes or aging. AGE administration represents an animal model system for the study of diabetic and aging complications as well as for assessing the efficacy of newly emerging therapies aimed at inhibiting advanced glycosylation.
Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). Much remains unknown about the basic biology of P. carinii and studies of this infection have been hampered by the lack of cultivation methods. We developed a sensitive and specific assay for P. carinii by utilizing DNA amplification of the P. carinii dihydrofolate reductase (DHFR) gene. By this method, P. carinii DNA was detected in the lungs of rats with experimentally induced P. carinii pneumonia 2 wk before the onset of histopathological changes. DNA amplification analysis of serum demonstrated that by 10 wk of corticosteroid treatment, 12 of 12 (100%) infected rats had circulating DHFR DNA. P. carinii DHFR DNA also was detected in the serum of patients with AIDS and active P. carinii pneumonia (12 of 14 sera collected prospectively). Patients with advanced AIDS but without a history of P. carinii pneumonia were negative by this assay (0 of 6 sera examined). Serum polymerase chain reaction may facilitate investigations into the natural history and epidemiology of P. carinii infection, provide insight into the pathogenesis of parasite dissemination, and offer a useful, noninvasive diagnostic test for the detection of human pneumocystosis.
Advanced glycosylation end products (AGEs) form spontaneously from glucose-derived Amadori products and accumulate on long-lived tissue proteins. AGEs have been implicated in the pathogenesis of several of the complications of aging and diabetes, including atherosclerosis and renal disease. With the use of recently developed AGE-specific antibodies, an AGE-modified form of human hemoglobin has been identified. Termed hemoglobin-AGE (Hb-AGE), this modified species accounts for 0.42 percent of circulating hemoglobin in normal individuals but increases to 0.75 percent in patients with diabetes-induced hyperglycemia. In a group of diabetic patients treated with the advanced glycosylation inhibitor aminoguanidine, Hb-AGE levels decreased significantly over a 1-month period. Hemoglobin-AGE measurements may provide an index of long-term tissue modification by AGEs and prove useful in assessing the contribution of advanced glycosylation to a variety of diabetic and age-related complications.
Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.
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