Showing papers by "Richard Bucala published in 2000"

Protection from septic shock by neutralization of macrophage migration inhibitory factor.

Abstract: Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis Experiments performed in TNFalpha knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation Anti-MIF antibody protected TNFalpha knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP Conversely, co-injection of recombinant MIF and E coli markedly increased the lethality of peritonitis Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention

Immunological Evidence that Non-carboxymethyllysine Advanced Glycation End-products Are Produced from Short Chain Sugars and Dicarbonyl Compounds in vivo

Abstract: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 cross-reacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. Non-CML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.

Neurotoxicity of advanced glycation end-products for cultured cortical neurons.

Abstract: The Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in aging. AGEs are believed also to contribute to the pathology of Alzheimer disease (AD) and other neurodegenerative processes. Incubation of cortical neurons with 5 immunochemically distinct AGEs, designated AGEs-1 to -5, produced a dose-dependent increase in neuronal cell-death, as assessed by MTT assay, Trypan blue and Hoechst 33258 staining. The structural epitope designated AGE-2 was found to have the greatest cytopathic effect and the neurotoxicity of AGE-2 was neutralized by the addition of an anti-AGE-2-specific antibody, but not by other types of anti-AGE antibodies. Distinct classes of AGE structures also have been established to circulate in the blood of individuals with diabetes mellitus and end-stage renal disease treated by hemodialysis (DM-HD). We fractionated serum from normal control and DM-HD patients by gel filtration and identified 2 fractions that contained AGE epitopes-1 to -5 and as well as the defined AGE structure carboxymethyllysine (CML). The addition of these 2 fractions led to the death of cultured neuronal cells and this cytotoxic effect was completely prevented by the addition of the anti-AGE-2-specific antibody. We propose that the structural epitope AGE-2 is an important toxic moiety for neuronal cells.

The proinflammatory mediator macrophage migration inhibitory factor induces glucose catabolism in muscle.

Abstract: Severe infection or tissue invasion can provoke a catabolic response, leading to severe metabolic derangement, cachexia, and even death. Macrophage migration inhibitory factor (MIF) is an important regulator of the host response to infection. Released by various immune cells and by the anterior pituitary gland, MIF plays a critical role in the systemic inflammatory response by counterregulating the inhibitory effect of glucocorticoids on immune-cell activation and proinflammatory cytokine production. We describe herein an unexpected role for MIF in the regulation of glycolysis. The addition of MIF to differentiated L6 rat myotubes increased synthesis of fructose 2,6-bisphosphate (F2,6BP), a positive allosteric regulator of glycolysis. Increased expression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) enhanced F2,6BP production and, consequently, cellular lactate production. The catabolic effect of TNF-alpha on myotubes was mediated by MIF, which served as an autocrine stimulus for F2, 6BP production. TNF-alpha administered to mice decreased serum glucose levels and increased muscle F2,6BP levels; pretreatment with a neutralizing anti-MIF mAb completely inhibited these effects. Anti-MIF also prevented hypoglycemia and increased muscle F2,6BP levels in TNF-alpha-knockout mice that were administered LPS, supporting the intrinsic contribution of MIF to these inflammation-induced metabolic changes. Taken together with the recent finding that MIF is a positive, autocrine stimulator of insulin release, these data suggest an important role for MIF in the control of host glucose disposal and carbohydrate metabolism.

Macrophage migration inhibitory factor release by macrophages after ingestion of Plasmodium chabaudi-infected erythrocytes: possible role in the pathogenesis of malarial anemia.

Abstract: Human falciparum malaria, caused by Plasmodium falciparum infection, results in 1 to 2 million deaths per year, mostly children under the age of 5 years. The two main causes of death are severe anemia and cerebral malaria. Malarial anemia is characterized by parasite red blood cell (RBC) destruction and suppression of erythropoiesis (the mechanism of which is unknown) in the presence of a robust host erythropoietin response. The production of a host-derived erythropoiesis inhibitor in response to parasite products has been implicated in the pathogenesis of malarial anemia. The identity of this putative host factor is unknown, but antibody neutralization studies have ruled out interleukin-1β, tumor necrosis factor alpha, and gamma interferon while injection of interleukin-12 protects susceptible mice against lethal P. chabaudi infection. In this study, we report that ingestion of P. chabaudi-infected erythrocytes or malarial pigment (hemozoin) induces the release of macrophage migration inhibitory factor (MIF) from macrophages. MIF, a proinflammatory mediator and counter-regulator of glucocorticoid action, inhibits erythroid (BFU-E), multipotential (CFU-GEMM), and granulocyte-macrophage (CFU-GM) progenitor-derived colony formation. MIF was detected in the sera of P. chabaudi-infected BALB/c mice, and circulating levels correlated with disease severity. Liver MIF immunoreactivity increased concomitant with extensive pigment and parasitized RBC deposition. Finally, MIF was elevated three- to fourfold in the spleen and bone marrow of P. chabaudi-infected mice with active disease, as compared to early disease, or of uninfected controls. In summary, the present results suggest that MIF may be a host-derived factor involved in the pathophysiology of malaria anemia.

De Novo Expression of Macrophage Migration Inhibitory Factor in Atherogenesis in Rabbits

Abstract: Macrophage migration inhibitory factor (MIF) has been shown to play an important role in macrophage-mediated diseases. We investigate the potential role of MIF in atherogenesis using a hypercholesterolemic rabbit model. New Zealand White rabbits fed with a 2% cholesterol diet developed hypercholesterolemia and early fatty streaks at 1 month. The lesions became advanced at 3 months and were associated with de novo MIF expression by vascular endothelial cells (VECs) and smooth muscle cells (SMCs), as demonstrated by immunohistochemistry, reverse transcriptase-polymerase chain reaction, and in situ hybridization. By contrast, there was no increase in MIF levels in rabbits fed a normal diet. In early atherogenesis, marked upregulation of MIF mRNA and protein by VECs and some intimal cells were closely associated with CD68(+) monocyte adhesion onto and subsequent migration into subendothelial space. Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, which may be associated with the macrophage-rich fatty streak lesion formation. Upregulation of MIF by SMCs is transient during atherogenesis. Importantly, strong MIF expression by activated macrophages may be responsible for the development of foam cell-rich lesions. Finally, the ability of MIF to induce intercellular adhesion molecule-1 expression by VECs implicates its pathogenic role in atherogenesis. In conclusion, the present study provides the first demonstration that MIF is markedly upregulated during atherogenesis. Upregulation of MIF by VECs and SMCs may play a role in macrophage adhesion, transendothelial migration, accumulation, and, importantly, transformation into foam cells. Furthermore, strong MIF expression by macrophages may both initiate and amplify the atherogenesis process.

Peripheral blood fibrocytes: mesenchymal precursor cells and the pathogenesis of fibrosis.

Abstract: Peripheral blood fibrocytes are a novel population of cells that rapidly enter sites of tissue injury and contribute to connective scar formation. Fibrocytes display a distinct cell surface phenotype (CD34+/CD45+/collagen I+), and are an abundant source of cytokines and growth factors that function to attract and activate inflammatory and connective tissue cells. Fibrocytes also are specialized to activate naive T cells against foreign antigen, and may play a critical role in the initiation of cognate immunity during the earliest phases of tissue injury. Recently, immunohistochemical studies of fibrotic tissues have shown that fibrocytes co-localize to areas of connective tissue matrix deposition. Peripheral blood fibrocytes thus may participate in the generation of excessive fibroses associated with various autoimmune disorders involving a persistent T-cell activation, as occurs in scleroderma.

Regulation of macrophage migration inhibitory factor by endogenous glucocorticoids in rat adjuvant-induced arthritis

Abstract: Objective To explore the regulation of macrophage migration inhibitory factor (MIF) by endogenous glucocorticoids in adjuvant-induced arthritis (AIA). Methods Adrenalectomy or sham operation was performed 2 days prior to adjuvant arthritis induction. Synovial explant supernatant levels of MIF and tumor necrosis factor α (TNFα) were measured by enzyme-linked immunosorbent assay (ELISA). Synovial MIF immunostaining was detected by 3-layer immunohistochemistry. Serum MIF levels were measured by Western blotting. Pituitary MIF release was measured by ELISA. Anti-MIF monoclonal antibody (mAb) or isotype-matched control antibody was administered to adrenalectomized (ADX) animals throughout AIA development. Results Compared with sham operation, adrenalectomy was associated with significant exacerbation of clinical disease parameters (P < 0.05). Adrenalectomy was associated with significantly reduced levels of synovial MIF, but not TNFα. In contrast, adrenalectomy was associated with increased serum MIF levels. Concomitant increased pituitary MIF levels were observed in ADX rats, consistent with the pituitary being the principal source of this increase. The administration of specific anti-MIF mAb conferred 100% protection from lethality during arthritis development and decreased arthritis disease expression. Conclusion These findings provide the first in vivo confirmation of the observation that endogenous glucocorticoids are involved in the regulation of MIF in a site of inflammation, and that local and systemic MIF production are differentially regulated in this setting. The reversal of disease in ADX rats by anti-MIF mAb suggests that balance between glucocorticoids and MIF may influence the expression of inflammatory disease.

Signal transduction. A most interesting factor.

Abstract: The effects of the signalling molecule MIF are quite well understood, but how it works remains a mystery. Some of the pathways behind its activity have now been revealed — with surprising results.

Involvement of Macrophage Migration Inhibitory Factor (MIF) in Experimental Uric Acid Nephropathy

Abstract: Deposition of uric acid in the kidney can lead to progressive tubulointerstitial injury with granuloma formation We hypothesized that uric acid crystal deposition may induce granuloma formation by stimulating local expression of macrophage migration inhibitory factor (MIF), which is a known mediator of delayed type hypersensitivity (DTH) A model of acute uric acid nephropathy was induced in rats by the administration of oxonic acid (an inhibitor of uricase), together with uric acid supplements MIF expression and local cellular response were examined by in situ hybridization and immunohistochemistry Kidney tissue examined at 35 days post-treatment showed widespread tubulointerstitial damage with intratubular uric acid crystal deposition and granuloma formation Tubules within the areas of granuloma showed a six-fold increase in MIF mRNA, compared with uninvolved areas by in situ hybridization Moreover, the areas of increased MIF mRNA expression correlated with sites of dense accumulation of macrophages and T cells, and these cells were activated when assessed by the expression of interleukin-2R (IL-2R) and (MHC) class II Interestingly, cytoplasmic staining for MIF protein in the uric acid (UA) crystal-associated granulomatous lesions was reduced, indicating a rapid MIF secretion by damaged tubules and macrophages secondary to uric acid crystal stimulation This was confirmed by the demonstration of a marked increase in urinary MIF protein by Western blot analysis Control rats fed either a normal diet or only oxonic acid had no discernible evidence of renal disease by routine light microscopy and minimal tubular expression of MIF mRNA and protein These data suggest that intrarenal granulomas in urate nephropathy may be the consequence of a crystal induced DTH reaction mediated by MIF

Compounds having mif antagonist activity

Abstract: There is disclosed a genus of optionally substituted Schiff base condensation products (and the carba analogs thereof) comprising an amino acid component and a benzaldehyde component, that have MIF (macrophage migration inhibitory factor) antagonist activity. The compounds are useful for treating a variety of diseases involving inflammatory activity or pro-inflammatory cytokine responses, such as autoimmune diseases, asthma, arthritis, EAE, ARDS and various forms of sepsis and septic shock, and other conditions characterized by underlying MIF responses including, for instance, tumor growth and neovascularization.

Method for determining MIF content

Abstract: The present invention relates to diagnostic methods for determining migration inhibitory factor (MIF) mRNA content in a sample, wherein MIF is human MIF polypeptide having a molecular weight of approximately 125 kDa and kits for detecting MIF