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Biomedical subjects

R Bucala

Publications and source records attributed to R Bucala.

At least 73 records · Page 4Linked to original sources
PubMed1999

Interaction of Borrelia burgdorferi with peripheral blood fibrocytes, antigen-presenting cells with the potential for connective tissue targeting.

BACKGROUND: Borrelia Burgdorferi has a predilection for collagenous tissue and can interact with fibronectin and cellular collagens. While the molecular mechanisms of how B. burgdorferi targets connective tissues and causes arthritis are not understood, the spirochetes can bind to a number of different cell types, including fibroblasts. A novel circulating fibroblast-like cell called the peripheral blood fibrocyte has recently been described. Fibrocytes express collagen types I and III as well as fibronectin. Besides playing a role in wound healing, fibrocytes have the potential to target to connective tissue and the functional capacity to recruit, activate, and present antigen to CD4(+) T cells. MATERIALS AND METHODS: Rhesus monkey fibrocytes were isolated and characterized by flow cytometry. B. burgdorferi were incubated with human or monkey fibrocyte cultures in vitro and the cellular interactions analyzed by light and electron microscopy. The two strains of B. burgdorferi studied included JD1, which is highly pathogenic for monkeys, and M297, which lacks the cell surface OspA and OspB proteins. RESULTS: In this study, we demonstrate that B. burgdorferi binds to both human and monkey (rhesus) fibrocytes in vitro. This process does not require OspA or OspB. In addition, the spirochetes are not phagocytosed but are taken into deep recesses of the cell membrane, a process that may protect them from the immune system. CONCLUSIONS: This interaction between B. burgdorferi and peripheral blood fibrocytes provides a potential explanation for the targeting of spirochetes to joint connective tissue and may contribute to the inflammatory process in Lyme arthritis.

Animals↗
PubMed1999

An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has been shown to counterregulate glucocorticoid action and to play an essential role in the activation of macrophages and T cells in vivo. MIF also may function as an autocrine growth factor in certain cell systems. We have explored the role of MIF in the growth of the 38C13 B cell lymphoma in C3H/HeN mice, a well-characterized syngeneic model for the study of solid tumor biology. MATERIALS AND METHODS: Tumor-bearing mice were treated with a neutralizing anti-MIF monoclonal antibody and the tumor response assessed grossly and histologically. Tumor capillaries were enumerated by immunohistochemistry and analyzed for MIF expression. The effect of MIF on endothelial cell proliferation was studied in vitro, utilizing both specific antibody and antisense oligonucleotide constructs. The role of MIF in angiogenesis also was examined in a standard Matrigel model of new blood vessel formation in vivo. RESULTS: The administration of anti-MIF monoclonal antibodies to mice was found to reduce significantly the growth and the vascularization of the 38C13 B cell lymphoma. By immunohistochemistry, MIF was expressed predominantly within the tumor-associated neovasculature. Cultured microvascular endothelial cells, but not 38C13 B cells, produced MIF protein and required its activity for proliferation in vitro. Anti-MIF monoclonal antibody also was found to markedly inhibit the neovascularization response elicited by Matrigel implantation. CONCLUSION: These data significantly expand the role of MIF in host responses, and suggest a new target for the development of anti-neoplastic agents that inhibit tumor neovascularization.

Animals↗
PubMed1998

Macrophage migration inhibitory factor expression in human renal allograft rejection.

BACKGROUND: Macrophage migration inhibitory factor (MIF) plays a pivotal role in immune-mediated diseases. Despite the long-standing association of MIF with the delayed-type hypersensitivity response, the potential role of MIF in allograft rejection is unknown. METHODS: MIF expression was assessed by in situ hybridization and immunohistochemistry staining in 62 biopsies of human renal allograft rejection and in normal human kidney. RESULTS: MIF mRNA and protein is constitutively expressed in normal kidney, being largely restricted to tubular epithelial cells, some glomerular epithelial cells, and vascular smooth muscle cells. In both acute and chronic renal allograft rejection, there was marked up-regulation of MIF mRNA and protein expression by intrinsic kidney cells such as tubular epithelial cells and vascular endothelial and smooth muscle cells. There was also MIF expression by infiltrating macrophages and T cells. Of note, macrophage and T cell infiltrates were largely restricted to areas with marked up-regulation of MIF expression, potentially contributing to the development of severe tubulitis and intimal or transmural arteritis. Quantitative analysis found that increased MIF expression in allograft rejection gave a highly significant correlation with macrophage and T cell accumulation in both the glomerulus and interstitium (P<0.001). In addition, the number of MIF+ tubules and interstitial MIF+ cells correlated significantly with the severity of allograft rejection (P<0.01), and the loss of renal function (P<0.01). In contrast, no up-regulation of renal MIF expression and no leukocyte accumulation was seen in allograft biopsies without evidence of rejection. CONCLUSIONS: This is the first study to demonstrate that local MIF expression is up-regulated during allograft rejection. The association between up-regulation of MIF expression, macrophage and T cell infiltration and the severity of renal allograft rejection suggests that MIF may be an important mediator in the process of allograft rejection.

Acute Disease↗
PubMed1998

Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of Gram-positive bacteria.

Discovered in the early 1960s as a T cell cytokine, the protein mediator known as macrophage migration inhibitory factor (MIF) has been found recently to be a pituitary peptide released during the physiological stress response, a proinflammatory macrophage cytokine secreted after LPS stimulation, and a T cell product expressed as part of the antigen-dependent activation response. We report herein that MIF also plays a critical role in the innate host response to staphylococcal and streptococcal exotoxins. In RAW 264.7 or elicited mouse peritoneal macrophages, peak MIF secretion was induced by concentrations of the staphylococcal toxic shock syndrome (TSS) toxin 1 (TSST-1) and the streptococcal pyrogenic exotoxin A as low as 10 pg/ml. Moreover, dose-response studies of splenocyte cytokine production showed that lower concentrations of TSST-1 (10 pg/ml) were needed to release MIF than to induce interleukin 2 or interferon-gamma secretion (1 ng/ml). We also studied the effect of neutralizing anti-MIF antibodies on TSST-1-induced lymphocyte proliferation and lethal toxic shock. Pretreatment of C57BL/6 mice with anti-MIF antibody 2 hr before TSST-1 injection prevented spleen enlargement and reduced by 50% the proliferation of splenocytes measured ex vivo. In a lethal mouse model of TSST-1-induced shock, anti-MIF antibody increased survival from 8% to 54% (P < 0.0001). These studies indicate that Gram-positive exotoxins are extremely potent inducers of MIF secretion and establish a critical role for MIF and the macrophage in the pathogenesis of the TSSs and in the innate immune response.

Animals↗
PubMed1998

Migration inhibitory factor induces killing of Leishmania major by macrophages: dependence on reactive nitrogen intermediates and endogenous TNF-alpha.

Macrophage migration inhibitory factor (MIF) is a product of activated T cells, anterior pituitary cells, and macrophages. MIF plays an important role in LPS-induced shock and delayed-type hypersensitivity. Furthermore, MIF exhibits a proinflammatory spectrum of action, promoting TNF-alpha production by macrophages, and counter-regulates glucocorticoid suppression of cytokine production. Here, we report that purified recombinant MIF activates murine macrophages to kill Leishmania major, with maximal effects at concentrations above 1 microg/ml. This MIF-mediated activation is specific, since it can be blocked completely by anti-MIF mAb. The MIF-mediated activation is dependent on TNF-alpha produced endogenously by macrophages, because the administration of anti-TNF-alpha antiserum markedly reduced the MIF effect. No MIF-mediated activation was observed in macrophages derived from TNF receptor p55 knockout mice, thus demonstrating the requirement of the smaller TNF receptor molecule for autocrine TNF-alpha signaling. A highly specific inhibitor of the inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl)lysine, dihydrochloride, also inhibited the action of MIF, suggesting an important role for iNOS in the antiparasitic properties of MIF. In line with this, no MIF-mediated activation was detected analyzing macrophages derived from iNOS-deficient mice. The effect of MIF was blocked completely by the macrophage-deactivating cytokines IL-10, IL-13, and TGF-beta. Finally, the expression of MIF mRNA and protein was up-regulated in lymph nodes of mice during the first week after infection with L. major. MIF therefore represents a cytokine involved not only in the recruitment of proinflammatory cells during infection but also in the complex regulation of the antimicrobial activity of these cells.

Animals↗
PubMed1998

Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products.

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

Antibodies, Monoclonal↗
PubMed1998

Human circulating eosinophils secrete macrophage migration inhibitory factor (MIF). Potential role in asthma.

Macrophage migration inhibitory factor (MIF) is a potent proinflammatory mediator that has been shown to potentiate lethal endotoxemia and to play a potentially important regulatory role in human acute respiratory distress syndrome (ARDS). We have investigated whether eosinophils are an important source of MIF and whether MIF may be involved in the pathophysiology of asthma. Unstimulated human circulating eosinophils were found to contain preformed MIF. Stimulation of human eosinophils with phorbol myristate acetate in vitro yielded significant release of MIF protein. For example, eosinophils stimulated with phorbol myristate acetate (100 nM, 8 h, 37 degreesC) released 1,539+/-435 pg/10(6) cells of MIF, whereas unstimulated cells released barely detectable levels (< 142 pg/10(6) cells, mean+/-SEM, n = 8). This stimulated release was shown to be (a) concentration- and time-dependent, (b) partially blocked by the protein synthesis inhibitor cycloheximide, and (c) significantly inhibited by the protein kinase C inhibitor Ro-31,8220. In addition, we show that the physiological stimuli C5a and IL-5 also cause significant MIF release. Furthermore, bronchoalveolar lavage fluid obtained from asthmatic patients contains significantly elevated levels of MIF as compared to nonatopic normal volunteers (asthmatic, 797.5+/-92 pg/ml; controls, 274+/-91 pg/ml). These results highlight the potential importance of MIF in asthma and other eosinophil-dependent inflammatory disorders.

Asthma↗
PubMed1998

Neuroimmunomodulation by macrophage migration inhibitory factor (MIF).

Recent studies have led to the discovery of a mediator that appears to act as an endogenous hormone to counterregulate glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, the structure of this protein was found to be that of macrophage migration inhibitory factor (MIF)--one of the first cytokines to be identified and described originally as a T lymphocyte factor that inhibited the random migration of macrophages. Macrophages and T cells release MIF in response to glucocorticoids, as well as upon activation by various proinflammatory stimuli. Once secreted MIF "overrides" the immunosuppressive effects of steroids on macrophage and T-cell cytokine production. MIF appears to fill an important gap in our understanding of how the host initiates and controls the immune response. Because glucocorticoids are an integral part of the host's metabolic "stress" response to infection or tissue invasion, the role of MIF is to act at an inflammatory site or lymph node to counterbalance the inhibitory effects of steroids on the immune response. Anti-MIF therapeutic strategies are under development and may prove to be a means to increase the immunosuppressive and anti-inflammatory properties of endogenously released glucocorticoids, thereby reducing the requirement for steroid therapy in a variety of autoimmune and inflammatory conditions.

Animals↗
PubMed1998

Advanced glycation end products increase retinal vascular endothelial growth factor expression.

Advanced glycation end products (AGEs) are linked with the development of diabetic retinopathy; however, the pathogenic mechanisms are poorly defined. Vascular endothelial growth factor (VEGF) levels are increased in ischemic and nonischemic diabetic retina, and VEGF is required for the development of retinal and iris neovascularization. Moreover, VEGF alone can induce much of the concomitant pathology of diabetic retinopathy. In this study, we found that AGEs increased VEGF mRNA levels in the ganglion, inner nuclear, and retinal pigment epithelial (RPE) cell layers of the rat retina. In vitro, AGEs increased VEGF mRNA and secreted protein in human RPE and bovine vascular smooth muscle cells. The AGE-induced increases in VEGF expression were dose- and time-dependent, inhibited by antioxidants, and additive with hypoxia. Use of an anti-VEGF antibody blocked the capillary endothelial cell proliferation induced by the conditioned media of AGE-treated cells. AGEs may participate in the pathogenesis of diabetic retinopathy through their ability to increase retinal VEGF gene expression.

Animals↗
PubMed1998

Role of cyclophilin A in the uptake of HIV-1 by macrophages and T lymphocytes.

Cyclophilins are a family of proteins that bind cyclosporin A (CsA) and possess peptidyl-prolyl cis-trans isomerase activity. In addition, they are secreted by activated cells and act in a cytokine-like manner, presumably via signaling through a cell surface cyclophilin receptor. More recently, host-derived cyclophilin A (CyPA) has been shown to be incorporated into HIV-1 virions and its incorporation essential for viral infectivity. Here we present evidence supporting a role for viral-associated CyPA in the early events of HIV-1 infection. We report that HIV-1 infection of primary peripheral blood mononuclear cells can be inhibited by: (i) an excess of exogenously added CyPA; (ii) a CsA analogue unable to enter the cells; (iii) neutralizing antibodies to CyPA. Taken together with our observations that recombinant CyPA-induced mobilization of calcium in immortalized, as well as primary, CD4+ T lymphocytes, and that incubation of T cells with iodinated CyPA, followed by chemical cross-linking, resulted in the formation of a high molecular mass complex on the cell surface, these results suggest that HIV-1-associated CyPA mediates an early event in viral infection via interaction with a cellular receptor. This interaction may present a target for anti-HIV therapies and vaccines.

Animals↗
PubMed1998

Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide.

The formation of amyloid within the islets of Langerhans is associated with the development of type II diabetes mellitus and occurs by the aggregation and insolubilization of islet amyloid polypeptide (IAPP). Recent in vitro studies suggest that amyloid formation follows a nucleation-dependent polymerization mechanism, i.e. aggregation is initiated by pre-formed aggregates or nucleation seeds. Modification of the Alzheimer's disease amyloid peptide by advanced glycosylation end products (AGEs), which form spontaneously by the non-enzymatic addition of glucose to protein amino groups, has been shown to enhance peptide aggregation in vitro. To explore the possibility that AGEs contribute to islet amyloid formation, we prepared AGE-modified IAPP (AGE-IAPP) in vitro and studied its properties by biochemical and biophysical techniques. AGE modification induced the formation of high-molecular-mass IAPP aggregates and amyloid formation was demonstrated by Congo red green-gold birefringence and by the presence of a characteristic fibrillar structure by electron microscopy. AGE-IAPP also showed an increase in cytotoxicity toward the astroglioma cell line HTB14. When added to soluble IAPP, AGE-IAPP seeds accelerated IAPP aggregation and abolished the nucleation period required for the polymerization of unseeded IAPP. Circular dichroism spectropolarimetry indicated that AGE-IAPP seeds may act as a template to stabilize the beta-sheet conformation of IAPP, thereby promoting its aggregation. Our studies demonstrate that AGE modification of IAPP results in high-molecular mass, fibrillar amyloid structures that nucleate IAPP amyloid formation and suggest a model for intra-islet amyloid deposition that may occur by the progressive advanced glycosylation of IAPP in vivo.

Amyloid↗
PubMed1998

Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.

We recently described a novel population of blood-borne cells, termed fibrocytes, that display a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and contribute to scar formation. To further characterize the role of these cells in vivo, we examined the expression of type I collagen and cytokine mRNAs by cells isolated from wound chambers implanted into mice. Five days after chamber implantation, CD34+ fibrocytes but not CD14+ monocytes or CD90+ T cells expressed mRNA for type I collagen. Fibrocytes purified from wound chambers also were found to express mRNA for IL-1beta, IL-10, TNF-alpha, JE/MCP, MIP-1alpha, MIP-1beta, MIP-2, PDGF-A, TGF-beta1, and M-CSF. The addition of IL-1beta (1-100 ng/ml), a critical mediator in wound healing, to fibrocytes isolated from human peripheral blood induced the secretion of chemokines (MIP-1alpha, MIP-1beta, MCP-1, IL-8, and GRO alpha), hemopoietic growth factors (IL-6, IL-10, and macrophage-CSF), and the fibrogenic cytokine TNF-alpha. By contrast, IL-1beta decreased the constitutive secretion of type I collagen as measured by ELISA. Additional evidence for a role for fibrocytes in collagen production in vivo was obtained in studies of livers obtained from Schistosoma japonicum-infected mice. Mouse fibrocytes localized to areas of granuloma formation and connective matrix deposition. We conclude that fibrocytes are an important source of cytokines and type I collagen during both the inflammatory and the repair phase of the wound healing response. Furthermore, IL-1beta may act on fibrocytes to effect a phenotypic transition between a repair/remodeling and a proinflammatory mode.

Animals↗
PubMed1998

Involvement of macrophage migration inhibitory factor in the evolution of rat adjuvant arthritis.

OBJECTIVE: Recent studies have established an essential role for macrophage migration inhibitory factor (MIF) in T cell and macrophage activation, both of which are characteristics of rat adjuvant arthritis. This study investigated the role of MIF in early adjuvant arthritis. METHODS: MIF was detected in rat synovium by immunohistochemistry and enzyme-linked immunosorbent assay using specific monoclonal antibodies (MAb). Anti-MIF MAb treatment was administered, and the effects on clinical aspects of adjuvant arthritis were assessed. RESULTS: MIF was absent from normal rat synovium prior to adjuvant injection, but was detectable on day 4 after injection (6 days before the onset of clinical disease) and was colocalized with ED-1+ macrophages throughout the evolution of the disease. Levels of MIF were increased in established adjuvant arthritis sera, and adjuvant arthritis synovial macrophages released MIF at a mean +/- SEM concentration of 607.9 +/- 201.5 pg/ml. Anti-MIF treatment led to profound, dose-dependent inhibition of the adjuvant arthritis clinical score, paw swelling, and synovial lavage leukocyte numbers (P < 0.001), and also resulted in reduced synovial macrophage and T cell accumulation. CONCLUSION: These findings demonstrate an important role for MIF in the evolution of rat adjuvant arthritis.

Animals↗
PubMed1998

Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features.

The classical T cell cytokine macrophage migration inhibitory factor (MIF) has reemerged recently as a critical mediator of the host immune and stress response. MIF has been found to be a mediator of several diseases including gram-negative septic shock and delayed-type hypersensitivity reactions. Its immunological functions include the modulation of the host macrophage and T and B cell response. In contrast to other known cytokines, MIF production is induced rather than suppressed by glucocorticoids, and MIF has been found to override the immunosuppressive effects of glucocorticoids. Recently, elucidation of the three-dimensional structure of MIF revealed that MIF has a novel, unique cytokine structure. Here the biological role of MIF is reviewed in view of its distinct immunological and structural properties.

Animals↗
PubMed1998

Extracellular matrix in human diabetic nephropathy: reduced expression of heparan sulphate in skin basement membrane.

In diabetic nephropathy, expression of glycosaminoglycan side chains of heparan sulphate proteoglycan in the glomerular basement membrane is reduced proportionally to the degree of proteinuria. We performed a cross-sectional study to evaluate whether non-vascular basement membranes also show a decrease in heparan sulphate side chain staining in patients with diabetic nephropathy. We evaluated the skin basement membrane for extracellular matrix components in the following groups: control subjects (n = 16); patients with Type 1 diabetes and normoalbuminuria (n = 17), microalbuminuria (n = 7), and macroalbuminuria (n = 16); patients with Type 1 diabetes and diabetic nephropathy undergoing renal replacement therapy (n = 13); and non-diabetic patients undergoing renal replacement therapy (n = 21). The following antibodies were used for this immunohistochemical study: monoclonal antibodies against the heparan sulphate side chain (JM403) and core protein (JM72) of the glomerular heparan sulphate proteoglycan; polyclonal antibodies against the core protein (B31); polyclonal antibodies against collagen types I, III, and IV, fibronectin, and laminin; and monoclonal antibodies against the noncollagenous domain of alpha1(collagen IV) and alpha3(collagen IV), against transforming growth factor beta(2G7), and against advanced glycosylation end products (4G9). Expression of heparan sulphate side chains was reduced in the skin basement membrane of patients with overt diabetic nephropathy, of those with Type 1 diabetes undergoing renal replacement therapy, and those with non-diabetic renal failure. Increased intensity of staining was found for collagen type I and advanced glycosylation end products in patients with diabetic nephropathy. Changes in the extracellular matrix of the skin basement membrane seem to be similar to those in the glomerular basement membrane. These findings support the suggestion that patients with diabetic nephropathy also have altered heparan sulphate and collagen staining in extrarenal basement membranes. However, patients with non-diabetic renal failure also had reduced expression of heparan sulphate in the skin basement membrane, suggesting that this finding is not specific for diabetic nephropathy.

Adult↗
PubMed1998

Aminoguanidine has an anti-atherogenic effect in the cholesterol-fed rabbit.

Advanced glycosylation endproducts (AGEs) which result from the non-enzymatic interaction of proteins and glucose are implicated in the vasculopathy of diabetes and aging. Since aminoguanidine (A) inhibits the accumulation of AGEs, we explored its effects on the development of atherosclerosis. Male New Zealand white cross rabbits fed a high cholesterol (1%) diet were randomized to control (C) or increasing doses of A treatment (25, 50 and 100 mg/kg A body weight). The animals were sacrificed after 12 weeks. Sudan IV was used to stain the lipid containing plaques of the aortic arch, thoracic and abdominal aorta and the surface area occupied by atheroma was assessed. Increasing doses of A treatment were associated with reduction in plaque formation in the aorta. At a dose of 100 mg/kg A, there was a 30, 49 and 48% reduction in plaque formation in the aortic arch, thoracic and abdominal aorta, respectively. There was a correlation between AGE levels and the degree of atheroma in these cholesterol fed rabbits (control, r = 0.75, P < 0.01; 100 mg/kg A, r = 0.59, P = 0.02). These data suggest that advanced glycation may participate in atherogenesis and raise the possibility that inhibitors of advanced glycation may retard this process.

Animals↗