Showing papers by "Richard Bucala published in 1996"

An essential regulatory role for macrophage migration inhibitory factor in T-cell activation

Abstract: The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses.

An agent cleaving glucose-derived protein crosslinks in vitro and in vivo

Abstract: GLUCOSE and other reducing sugars react with proteins by a non-enzymatic, post-translational modification process called non-enzymatic glycosylation or glycation. The sugar-derived carbonyl group adds to a free amine, forming a reversible adduct which over time rearranges to produce a class of products termed advanced-glycation end-products (AGEs). These remain irreversibly bound to macromolecules and can covalently crosslink proximate amino groups1,2. The formation of AGEs on long-lived connective tissue and matrix components accounts largely for the increase in collagen crosslinking that accompanies normal ageing and which occurs at an accelerated rate in diabetes3,4. AGEs can activate cellular receptors and initiate a variety of pathophysiological responses5–9. They modify an appreciable fraction of circulating low-density lipoproteins preventing uptake of these particles by their high-affinity tissue receptors10,11. Advanced glycation has also been implicated in the pathology of Alzheimer's disease12,13. Because AGEs may form by a pathway involving reactive α-dicarbonyl intermediates1,2,14, we investigated a potential pharmacological strategy for selectively cleaving the resultant glucose-derived protein crosslinks. We now describe a prototypic AGE crosslink 'breaker', N-phenacylthiazolium bromide (PTB), which reacts with and cleaves covalent, AGE-derived protein crosslinks. The ability of PTB to break AGE crosslinks in vivo points to the importance of an α-dicarbonyl intermediate in the advanced glycation pathway and offers a potential therapeutic approach for the removal of established AGE crosslinks.

The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction.

Abstract: Recent studies of melanin biosynthesis have uncovered an unusual enzymatic activity which converts the non-naturally occurring D-isomer of 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The aim of the present investigation was to isolate and characterize the enzyme catalyzing this tautomerization reaction. After we performed a tissue survey of D-dopachrome tautomerase activity, 10 bovine lenses were homogenized and used as a source of enzyme. A soluble fraction was obtained by high-speed centrifugation and subjected to successive FPLC chromatography on Phenyl-sepharose, Mono S cation-exchange, and Superdex gel-filtration. The isolated enzyme was electrophoresed, blotted onto PVDF membrane, and the N terminus analyzed by gas phase micro-sequencing. The protein catalyzing the conversion of D-dopachrome to DHICA was purified to homogeneity in 14% yield and showed a molecular weight of 12 kD when analyzed by SDS-PAGE. The first 27 amino acid residues of this protein were sequenced and found to be identical with those of bovine macrophage migration inhibitory factor (MIF). The catalytic activity of native MIF was confirmed by studies of purified recombinant human MIF, which showed the same tautomerase activity. While L-dopachrome was not a substrate for this reaction, the methyl esters of the L- and D-isomers were found to be better substrates for MIF than D-dopachrome. MIF has been described recently to be an anterior pituitary hormone and to be released from immune cells stimulated by low concentrations of glucocorticoids. Once secreted, MIF acts to control, or counter-regulate, the immunosuppressive effects of glucocorticoids on the immune system. Although the tested substrate, D-dopachrome, does not occur naturally, the observation that MIF has tautomerase activity suggests that MIF may mediate its biological effects by an enzymatic reaction. These data also offer a potential approach for the design of small molecule pharmacological inhibitors of MIF that may modulate its potent immunoregulatory effects in vivo.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Abstract: Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction

Abstract: 30 years ago, investigations into the molecular basis of the delayed-type hypersensitivity reaction (DTH) provided evidence for the first lymphokine activity: a lymphocyte-derived mediator called macrophage migration inhibitory factor (MIF), which inhibited the random migration of peritoneal macrophages. Despite the long-standing association of MIF with the DTH reaction and the cloning of a human protein with macrophage migration inhibitory activity, the precise role of MIF in this classic cell-mediated immune response has remained undefined. This situation has been further complicated by the fact that two other cytokines, interferon gamma and IL-4, similarly inhibit macrophage migration and by the identification of mitogenic contaminants in some preparations of cloned human MIF. Using recently developed molecular probes for mouse MIF, we have examined the role of this protein in a classical model of DTH, the tuberculin reaction in mice. Both MIF messenger RNA and protein were expressed prominently in DTH lesions, as assessed by reverse transcription polymerase chain reaction, in situ hybridization, and immunostaining with anti-MIF antibody. The predominant cellular origin of MIF appeared to be the monocyte/macrophage, a cell type identified recently to be a major source of MIF release in vivo. The administration of neutralizing anti-MIF antibodies to mice inhibited significantly the development of DTH, thus affirming the central role of MIF in this classic immunological response.

MIF rediscovered: cytokine, pituitary hormone, and glucocorticoid-induced regulator of the immune response.

Abstract: The protein that has been historically called macrophage migration inhibitory factor (MIF) was one of the first cytokine activities to be discovered and was originally described to be a T lymphocyte product that inhibited the random migration of macrophages. Over the years, additional molecules with MIF "activity" have been described and the precise role of the original MIF "protein" remained enigmatic. Recent studies have led to the discovery of a pituitary mediator that appears to act as the counterregulatory hormone for glucocorticoid action within the immune system. Isolated as a product of murine anterior pituitary cells, this peptide was sequenced and found to be the mouse homolog of MIF. MIF has the unique property of being released from macrophages and T cells in response to physiological concentrations of glucocorticoids. The secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once released, MIF "overrides" or counterregulates the immunosuppressive effects of steroids on immune cell activation and cytokine production. These observations suggest that MIF fills an important gap in our understanding of how the host initiates and controls immunity. Because glucocorticoids are an integral part of the host's global response to infection or tissue invasion, the physiological role of MIF is to act at an inflammatory site or lymph node to counterbalance the profound inhibitory effects of steroids on the immune response.

Prevention of cardiovascular and renal pathology of aging by the advanced glycation inhibitor aminoguanidine.

Abstract: Human aging is impacted severely by cardiovascular disease and significantly but less overtly by renal dysfunction. Advanced glycation endproducts (AGEs) have been linked to tissue damage in diabetes and aging, and the AGE inhibitor aminoguanidine (AG) has been shown to inhibit renal and vascular pathology in diabetic animals. In the present study, the effects of AG on aging-related renal and vascular changes and AGE accumulation were studied in nondiabetic female Sprague-Dawley (S-D) and Fischer 344 (F344) rats treated with AG (0.1% in drinking water) for 18 mo. Significant increases in the AGE content in aged cardiac (P < 0.05), aortic (P < 0.005), and renal (P < 0.05) tissues were prevented by AG treatment (P < 0.05 for each tissue). A marked age-linked vasodilatory impairment in response to acetylcholine and nitroglycerine was prevented by AG treatment (P < 0.005), as was an age-related cardiac hypertrophy evident in both strains (P < 0.05). While creatinine clearance was unaffected by aging in these studies, the AGE/ creatinine clearance ratio declined 3-fold in old rats vs. young rats (S-D, P < 0.05; F344, P < 0.01), while it declined significantly less in AG-treated old rats (P < 0.05). In S-D but not in F344 rats, a significant (P < 0.05) age-linked 24% nephron loss was completely prevented by AG treatment, and glomerular sclerosis was markedly suppressed (P < 0.01). Age-related albuminuria and proteinuria were markedly inhibited by AG in both strains (S-D, P < 0.01; F344, P < 0.01). These data suggest that early interference with AGE accumulation by AG treatment may impart significant protection against the progressive cardiovascular and renal decline afflicting the last decades of life.

Recent progress in advanced glycation and diabetic vascular disease: role of advanced glycation end product receptors.

Abstract: Advanced glycosylation end products (AGEs) form principally from the rearrangement of early glycation products, i.e., Amadori products, which produce a class of stable moieties that possess distinctive chemical crosslinking and biological properties. It has been generally believed that proteins with half-lives of longer than a few weeks are most susceptible to advanced glycosylation and that the highest levels of AGEs occur on proteins that comprise the long-lived structural components of connective tissue matrix and basement membrane.

De novo renal expression of macrophage migration inhibitory factor during the development of rat crescentic glomerulonephritis

Abstract: Macrophage migration inhibitory factor (MIF), a key mediator of the delayed-type hypersensitivity response, was originally thought to be produced by activated T cells However, recent studies have found that MIF is produced in many cell types including monocytes/macrophages and anterior pituitary cells The current study has examined MIF expression in normal and diseased kidney using in situ hybridization, immunohistochemistry, and Northern blotting MIF mRNA and protein are constitutively expressed in normal kidney, being largely restricted to tubular epithelial cells and some glomerular visceral and parietal epithelial cells During the development of rat anti-glomerular basement membrane glomerulonephritis, a model of macrophage-mediated renal injury, there was marked de novo expression of MIF by intrinsic kidney cells including endothelium and glomerular and tubular epithelial cells Up-regulation of MIF expression correlated with macrophage accumulation within the glomerulus (P < 0001) and tubulointerstitium (P < 0001) Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, contributing to focal glomerular and tubulointerstitial lesion formation In addition, up-regulation of MIF expression by parietal epithelial cells was associated with macrophage accumulation within Bowman's space and crescent formation Combined in situ hybridization and immunostaining also demonstrated MIF expression by macrophages, T cells, and fibroblast-like cells within renal lesions In conclusion, these data provide the first demonstration that renal epithelial cells are a major source of MIF in both normal and diseased kidney Furthermore, the up-regulation of MIF expression may play an important role in macrophage accumulation and progressive renal injury in rat crescentic glomerulonephritis

The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer

Abstract: The subunit structure of human macrophage migration inhibitory factor (MIF) has been studied by preliminary X-ray analysis of wild-type and selenomethionine-MIF and dynamic light scattering. Crystal form I of MIF belongs to space group P2(1)2(1)2(1) and is grown from 2 M ammonium sulfate at pH 8.5. A native data set has been collected to 2.4 A resolution. Self-rotation studies and Van values indicate that three molecules per asymmetric unit are present. A data set to 2.8 A resolution has been collected for crystal form II, which belongs to space group P3(1)21 or P3(2)21 and grows from 2 M ammonium sulfate, 2% polyethylene glycol (average molecular mass 400) 0.1 M HEPES, pH 7.5. Three, four, five or six monomers in the asymmetric unit are consistent with Van values for this crystal form. Analysis of crystal form II containing selenomethionine-MIF indicates nine selenium sites are present per asymmetric unit. Dynamic light scattering of MIF suggests that the major form of the protein in solution is a trimer. The results of these studies are in contrast to previous reports indicating that MIF is a monomer or dimer. The subunit arrangement of MIF is similar to that of tumor necrosis factor and suggests that signal transduction might require trimerization of receptor subunits.

The role of advanced glycosylation end-products in the pathogenesis of atherosclerosis

Abstract: Coronary artery disease and cerebrovascular disease due to the rapid progression of atherosclerosis is the principal cause of death in diabetes mellitus. Modification of low-density lipoproteins (LDL) by advanced glycosylation end-products (AGE) may play a central role in the development of atherosclerosis, especially in diabetic patients. An AGE-modified form of LDL (AGE-LDL) has been found to circulate in human plasma, and AGE modifications have been identified as being present on both the apoprotein (ApoB) and the phospholipid components of LDL. By utilizing an AGE-specific ELISA, we measured the AGE attached to the ApoB and lipid components of LDL from normal controls and diabetic patients with or without end-stage renal disease (ESRD), as well as lipid oxidation. AGE-ApoB, AGE-lipid and oxidized LDL (Ox-LDL) in diabetic patients were significantly higher than those in patients without diabetes. The correlation between AGE-ApoB and AGE-lipid were highly significant. An especially marked elevation of AGE-LDL was found in diabetic patients with ESRD. The correlation between the serum total cholesterol and the AGE-LDL (AGE-ApoB and AGE-lipid) was significant. In addition, based on the known biological properties of AGE-modified peptide (AGE-peptide), we have proposed that these chemically reactive circulating AGE-peptides contribute to tissue injury by reattaching to susceptible target proteins both within and outside the vasculature, and that this process accelerates vascular pathology in diabetic patients. These data indicate that AGE-modified LDLs may represent a particularly atherogenic form of LDL, and AGE-LDLs as well as AGE-peptides are likely to contribute to the development of atherosclerosis in diabetic patients.

Site-specific modification of apolipoprotein B by advanced glycosylation end-products: implications for lipoprotein clearance and atherogenesis

Abstract: An AGE-modified form of LDL (AGE-LDL) circulates in patients with diabetes mellitus or renal insufficiency and shows impaired plasma clearance kinetics when injected into transgenic mice that express the human LDL receptor. The advanced glycosylation inhibitor aminoguanidine decreases plasma LDL in diabetic patients, further suggesting that the AGE modification of LDL contributes significantly to increased LDL in vivo. We utilized AGE-specific antibodies to identify the major site(s) of AGE modification within apolipoprotein B (apoB), which impairs the binding of AGE-LDL by human fibroblast LDL receptors. Despite the large size of apoB (4536 amino acid residues) and its high content of potentially reactive lysines, the predominant site of AGE-immunoreactivity was found to lie within a single 67 amino acid domain located 1791 residues N-terminal to the LDL receptor binding site. These data point to the high specificity and reactivity of this site toward AGE formation and to a significant structural interaction between this region of apoB and the LDL receptor binding domain. A low but detectable degree of AGE modification was found to affect this site in LDL which was isolated from normal, non-diabetic individuals, suggesting that advanced glycosylation may contribute to elevated LDL in the general population as well.

Delayed-type hypersensitivity mediates Bowman's capsule rupture in Tamm—Horsfall protein-induced tubulointerstitial nephritis in the rat

Abstract: Summary: It is generally thought that disruption of Bowman's capsule, a feature of progressive forms of glomerulonephritis, is mediated by leucocytes which accumulate within Bowman's space. However, we have recently shown that periglomerular T cell and macrophage accumulation is associated with Bowman's capsule rupture irrespective of crescent formation in rat crescentic glomerulonephritis. Thus, to determine whether periglomerular leucocytes can mediate rupture of Bowman's capsule, we have examined Bowman's capsule integrity in a model of immune cell-mediated interstitial nephritis which is devoid of glomerular inflammation. Tubulointerstitial nephritis was induced in rats by repeated immunization with autologous Tamm-Horsfall protein (THP) or adjuvant alone (control) and groups of animals were killed on weeks 26, 30 and 34. All animals immunized with autologous THP developed a humoral immune response to the antigen as measured by serum antibodies. In addition, a cellular immune response to autologous THP was demonstrated by a skin delayed-type hypersensitivity (DTH) response and prominent interstitial T cell and macrophage infiltration around THP+ tubules. Marked periglomerular leucocyte infiltration developed around some glomeruli in association with granular THP deposition and increased local expression of intercellular adhesion molecule-1 (ICAM-1) and the classic DTH associated cytokine macrophage migration inhibitory factor. On week 26 of the disease, disruption of Bowman's capsule was evident in 5.5 ± 2.8% of glomeruli and this increased to 9.5 ± 4.8% of glomeruli by week 34. Disruption of Bowman's capsule integrity was invariably associated with THP deposition and a significant periglomerular infiltrate of T cells and macrophages, whereas glomeruli with little periglomerular infiltration retained an intact Bowman's capsule. Strong lysozyme staining of macrophage-like cells at sites of rupture suggest that degradation of Bowman's capsule was mediated by macrophages through the production of proteolytic enzymes. In conclusion, these results demonstrate that periglomerular leucocytes can cause Bowman's capsule rupture through a DTH mechanism.