Showing papers by "Richard Bucala published in 2012"

Fibrocytes in Health and Disease

Abstract: Fibrocytes, a group of bone marrow-derived mesenchymal progenitor cells, were first described in 1994 as fibroblast-like, peripheral blood cells that migrate to regions of tissue injury. These cells are unique in their expression of extracellular matrix proteins concomitantly with markers of hematopoietic and monocyte lineage. The involvement of fibrocytes and the specific role they play in the process of wound repair has been a focus of study since their initial description. Fibrocytes contribute to the healing repertoire via several mechanisms; they produce a combination of cytokines, chemokines, and growth factors to create a milieu favorable for repair to occur; they serve as antigen presenting cells (APCs); they contribute to wound closure; and, they promote angiogenesis. Furthermore, regulatory pathways involving serum amyloid P, leukocyte-specific protein 1, and adenosine A2A receptors have emphasized the significant role that fibrocytes have in wound healing and fibrosis. The therapeutic targeting of fibrocytes holds promise for the augmentation of wound repair and the treatment of different fibrosing disorders.

Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly.

Abstract: Aging is accompanied by a progressive decline in immune function. Studies have shown age-related decreases in the expression and signaling efficiency of Toll-like receptors (TLRs) in monocytes and dendritic cells and dysregulation of macrophage TLR3. Using a multivariable mixed effect model, we report a highly significant increase in TLR5-induced production of IL-8 from monocytes of older individuals (P < 0.0001). Elevated IL-8 is accompanied by increased expression of TLR5, both protein and mRNA, and by increased levels of TLR5-mediated phosphorylation of MAPK p38 and ERK. We noted incomplete activation of NF-κB in response to TLR5 signaling in monocytes of elderly donors, as reflected by the absence of an associated increase in the production of TNF-α. Elevated TLR5 may provide a critical mechanism to enhance immune responsiveness in older individuals.

Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise

Abstract: Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif−/− mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.

Small molecule inhibition of 6-phosphofructo-2-kinase suppresses t cell activation.

Abstract: T cell activation is associated with a rapid increase in intracellular fructose-2,6-bisphosphate (F2,6BP), an allosteric activator of the glycolytic enzyme, 6-phosphofructo-1-kinase. The steady state concentration of F2,6BP in T cells is dependent on the expression of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and the fructose-2,6-bisphosphatase, TIGAR. Of the PFKFB family of enzymes, PFKFB3 has the highest kinase:bisphosphatase ratio and has been demonstrated to be required for T cell proliferation. A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), recently has been shown to reduce F2,6BP synthesis, glucose uptake and proliferation in transformed cells. We hypothesized that the induction of PFKFB3 expression may be required for the stimulation of glycolysis in T cells and that exposure to the PFKFB3 antagonist, 3PO, would suppress T cell activation. We examined PFKFB1-4 and TIGAR expression and F2,6BP concentration in purified CD3+ T cells stimulated with microbead-conjugated agonist antibodies specific for CD3 and the co-stimulatory receptor, CD28. We then determined the effect of 3PO on anti-CD3/anti-CD28-induced T cell activation, F2,6BP synthesis, 2-[1-14C]-deoxy-d-glucose uptake, lactate secretion, TNF-α secretion and proliferation. Finally, we examined the effect of 3PO administration on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice. We found that purified human CD3+ T cells express PFKFB2, PFKFB3, PFKFB4 and TIGAR, and that anti-CD3/anti-CD28 conjugated microbeads stimulated a >20-fold increase in F2,6BP with a coincident increase in protein expression of the PFKFB3 family member and a decrease in TIGAR protein expression. We then found that exposure to the PFKFB3 small molecule antagonist, 3PO (1–10 μM), markedly attenuated the stimulation of F2,6BP synthesis, 2-[1-14C]-deoxy-D-glucose uptake, lactate secretion, TNF-α secretion and T cell aggregation and proliferation. We examined the in vivo effect of 3PO on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice and found that 3PO suppressed the development of both T cell-dependent models of immunity in vivo. Our data demonstrate that inhibition of the PFKFB3 kinase activity attenuates the activation of T cells in vitro and suppresses T cell dependent immunity in vivo and indicate that small molecule antagonists of PFKFB3 may prove effective as T cell immunosuppressive agents.

A Plasmodium-encoded cytokine suppresses T-cell immunity during malaria

Abstract: The inability to acquire protective immunity against Plasmodia is the chief obstacle to malaria control, and inadequate T-cell responses may facilitate persistent blood-stage infection. Malaria is characterized by a highly inflammatory cytokine milieu, and the lack of effective protection against infection suggests that memory T cells are not adequately formed or maintained. Using a genetically targeted strain of Plasmodium berghei, we observed that the Plasmodium ortholog of macrophage migration inhibitory factor enhanced inflammatory cytokine production and also induced antigen-experienced CD4 T cells to develop into short-lived effector cells rather than memory precursor cells. The short-lived effector CD4 T cells were more susceptible to Bcl-2–associated apoptosis, resulting in decreased CD4 T-cell recall responses against challenge infections. These findings indicate that Plasmodia actively interfere with the development of immunological memory and may account for the evolutionary conservation of parasite macrophage migration inhibitory factor orthologs.

The Cytokine Midkine and Its Receptor RPTPζ Regulate B Cell Survival in a Pathway Induced by CD74

Abstract: Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.

Role of Macrophage Migration Inhibitory Factor in the Regulatory T Cell Response of Tumor-Bearing Mice

Abstract: Macrophage migration inhibitory factor (MIF) is involved in tumorigenesis by facilitating tumor proliferation and evasion of apoptosis; however, its role in tumor immunity is unclear. In this study, we investigated the effect of MIF on the progression of the syngenic, CT26 colon carcinoma and the generation of tumor regulatory T cells (Tregs). The results showed that the tumor growth rate was significantly lower in MIF knockout (MIF(-/-)) mice than in wild-type (MIF(+/+)) mice. Flow cytometric analysis of both spleen and tumor cells revealed that MIF(-/-) mice had significantly lower levels of tumor-associated CD4(+)Tregs than MIF(+/+) mice. The splenic cells of MIF(-/-) mice also showed a decrease in CD8(+)Tregs, which was accompanied by an increase in CD8-induced tumor cytotoxicity. Interestingly, the inducible Treg response in spleen cells to anti-CD3/CD28 plus IL-2 plus TGF-β was greater in MIF(-/-) mice than in MIF(+/+) mice. Spleen cells of MIF(-/-) mice, stimulated with anti-CD3/CD28, produced lower levels of IL-2, but not TGF-β, than those of MIF(+/+) mice, which was recovered by the addition of recombinant MIF. Conversely, a neutralizing anti-MIF Ab blocked anti-CD3-induced IL-2 production by splenocytes of MIF(+/+) mice and suppressed the inducible Treg generation. Moreover, the administration of IL-2 into tumor-bearing MIF(-/-) mice restored the generation of Tregs and tumor growth. Taken together, our data suggest that MIF promotes tumor growth by increasing Treg generation through the modulation of IL-2 production. Thus, anti-MIF treatment might be useful in enhancing the adaptive immune response to colon cancers.

Macrophage Migration Inhibitory Factor Enzymatic Activity, Lung Inflammation, and Cystic Fibrosis

Abstract: Rationale: Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator with unique tautomerase enzymatic activity; the precise function has not been clearly defined. We previously demonstrated that individual patients with cystic fibrosis (CF) who are genetically predisposed to be high MIF producers develop accelerated end-organ injury.Objectives: To characterize the effects of the MIF-CATT polymorphism in patients with CF ex vivo. To investigate the role of MIF’s tautomerase activity in a murine model of Pseudomonas aeruginosa infection.Methods: MIF and tumor necrosis factor (TNF)-α protein levels were assessed in plasma or peripheral blood mononuclear cell (PBMC) supernatants by ELISA. A murine pulmonary model of chronic Pseudomonas infection was used in MIF wild-type mice (mif+/+) and in tautomerase-null, MIF gene knockin mice (mif P1G/P1G).Measurements and Main Results: MIF protein was measured in plasma and PBMCs from 5- and 6-CATT patients with CF; LPS-induced TNF-α production from PB...

MIF Produced by Bone Marrow–Derived Macrophages Contributes to Teratoma Progression after Embryonic Stem Cell Transplantation

Abstract: Although stem cell therapy holds promise as a potential treatment in a number of diseases, the tumorigenicity of embryonic stem cells (ESC) and induced pluripotent stem cells remains a major obstacle. In vitro predifferentiation of ESCs can help prevent the risk of teratoma formation, yet proliferating neural progenitors can generate tumors, especially in the presence of immunosuppressive therapy. In this study, we investigated the effects of the microenvironment on stem cell growth and teratoma development using undifferentiated ESCs. Syngeneic ESC transplantation triggered an inflammatory response that involved the recruitment of bone marrow(BM)–derivedmacrophages.ThesemacrophagesdifferentiatedintoanM2orangiogenicphenotypethat expressed multiple angiogenic growth factors and proteinases, such as macrophage migration inhibitory factor (MIF), VEGF, and matrix metalloproteinase 9, creating a microenvironment that supported the initiation of teratoma development. Genetic deletion of MIF from the host but not from ESCs specifically reduced angiogenesis and teratoma growth, and MIF inhibition effectively reduced teratoma development after ESC transplantation. Together, our findings show that syngeneic ESC transplantation provokes an inflammatory response that involves the rapid recruitment and activation of BM-derived macrophages, which may be a crucial driving force in the initiation and progression of teratomas. Cancer Res; 72(11); 2867–78. � 2012 AACR.

The outcome of renal ischemia-reperfusion injury is unchanged in AMPK-β1 deficient mice.

Abstract: Aim Activation of the master energy-regulator AMP-activated protein kinase (AMPK) in the heart reduces the severity of ischemia-reperfusion injury (IRI) but the role of AMPK in renal IRI is not known. The aim of this study was to determine whether activation of AMPK by acute renal ischemia influences the severity of renal IRI. Methods AMPK expression and activation and the severity of renal IRI was studied in mice lacking the AMPK β1 subunit and compared to wild type (WT) mice. Results Basal expression of activated AMPK, phosphorylayed at αThr172, was markedly reduced by 96% in AMPK-β1−/− mice. Acute renal ischaemia caused a 3.2-fold increase in α1-AMPK activity and a 2.5-fold increase in α2-AMPK activity (P<0.001) that was associated with an increase in AMPK phosphorylation of the AMPK-α subunit at Thr172 and Ser485, and increased inhibitory phosphorylation of the AMPK substrate acetyl-CoA carboxylase. After acute renal ischemia AMPK activity was reduced by 66% in AMPK-β1−/− mice compared with WT. There was no difference, however, in the severity of renal IRI at 24-hours between AMPK-β1−/− and WT mice, as measured by serum urea and creatinine and histological injury score. In the heart, macrophage migration inhibitory factor (MIF) released during IRI contributes to AMPK activation and protects from injury. In the kidney, however, no difference in AMPK activation by acute ischemia was observed between MIF−/− and WT mice. Compared with the heart, expression of the MIF receptor CD74 was found to be reduced in the kidney. Conclusion The failure of AMPK activation to influence the outcome of IRI in the kidney contrasts with what is reported in the heart. This difference might be due to a lack of effect of MIF on AMPK activation and lower CD74 expression in the kidney.

Virtual screening and optimization yield low-nanomolar inhibitors of the tautomerase activity of Plasmodium falciparum macrophage migration inhibitory factor.

Abstract: The Plasmodium falciparum orthologue of the human cytokine, macrophage migratory inhibitory factor (PfMIF), is produced by the parasite during malaria infection and modulates the host's immune response. As for other MIF orthologues, PfMIF has tautomerase activity, whose inhibition may influence the cytokine activity. To identify small-molecule inhibitors of the tautomerase activity of PfMIF, virtual screening has been performed by docking 2.1 million compounds into the enzymatic site. Assaying of 17 compounds identified four as active. Substructure search for the most potent of these compounds, a 4-phenoxypyridine analogue, identified four additional compounds that were purchased and also shown to be active. Thirty-one additional analogues were then designed, synthesized, and assayed. Three were found to be potent PfMIF tautomerase inhibitors with K(i) of ∼40 nM; they are also highly selective with K(i) > 100 μM for human MIF.

Fibrocytes in Fibrotic Diseases and Wound Healing

Abstract: Background: First described in 1994, fibrocytes are now appreciated to participate in different inflammatory and fibrogenic processes as well as in wound healing. Fibrocytes are unique in their expression of both myeloid and connective tissue products, which include a distinct cytokine and surface marker expression profile. Recent studies suggest their clinical utility as predictive biomarkers and as targets for therapeutic intervention. The Problem: Fibrocytes are involved in physiological and beneficial processes such as wound repair. Their involvement in detrimental processes such as aberrant collagen deposition in different fibrosing diseases reveals the sensitive balance in which these bone marrow progenitors have to be maintained. Basic/Clinical Science Advances: The enumeration of circulating fibrocytes and correlation with clinical severity of different fibrosing disorders is one of the most promising advantages in recent fibrocyte research. Besides their potential as a biomarker, fibrocytes may b...

Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease

Abstract: Scleroderma is an autoimmune disease characterized by the progressive and dysregulated accumulation of collagen in the skin and internal organs. Pulmonary complications including interstitial lung disease have emerged as the greatest cause of mortality in this disease. Because treatments are limited, new areas of investigation are sorely needed. An emerging area of interest in this field is a potential role for fibrocytes as biomarkers or mediators of disease. Fibrocytes are monocyte-derived mesenchymal progenitor cells that exhibit features of extracellular matrix production and wound contraction in addition to immunologic functions such as cytokine and chemokine production, antigen presentation, leukocyte trafficking, and modulation of angiogenesis. Fibrocytes could participate in the pathogenesis of scleroderma lung disease through any or all of these functions and may be useful biomarkers of disease activity. This chapter presents protocols that have been developed for the study of fibrocytes obtained from human circulation and tissues. Protocols for the quantification of fibrocytes in murine models also are described, along with discussion of common technical challenges. It is hoped that this information will allow further investigation of the role that fibrocytes might play in Scleroderma-related lung disease and perhaps lead to new areas of study in this difficult-to-treat and deadly disease.

Fibrocytes in rheumatoid pannus: seed and soil?

Abstract: Current views of rheumatoid arthritis (RA) pathogenesis emphasize an autoimmune etiology marshalling components of the innate and adaptive immune responses (1). As a consequence of rheumatoid inflammation, joint synovium develops into the erosive granulation tissue known as pannus, which is a pathologic hallmark of RA (2). Within pannus tissue, there is an enormous increase in the number of fibroblast-like synoviocytes, persistent inflammatory foci, lymphoid germinal centers, and angiogenesis. In vitro studies have established that fibroblasts isolated from the synovia of patients with RA exhibit an activated phenotype with enhanced proliferation, invasive properties, and increased secretion of growth-promoting cytokines and matrix-degrading enzymes. Rheumatoid synovial fibroblasts proliferate in an anchorage-independent manner, escape contact inhibition, and invade cartilage implants (2–5). Remarkably, this aggressive phenotype endures after multiple cell passages and prolonged culture, suggesting that synovial fibroblasts are persistently, if not irreversibly, altered in the rheumatoid joint. The precise manner in which synovial fibroblasts maintain this activated state after removal from the inflammatory articular milieu is unknown, but such maintenance may result from a selfperpetuating or reentrant stimulatory pathway involving autocrine mediators and sustained intracellular signaling. There is evidence in rheumatoid synovial fibroblasts for protooncogene activation and for the functional (and perhaps mutational) inactivation of the tumor suppressor gene p53, which regulates cell cycle progression (6). Although the notion of a mutational origin for pannus tissue has been largely discounted (and does not account for multicentric rheumatoid synovitis), longterm epigenetic changes may explain the activated phenotype of rheumatoid synovial fibroblasts. Beyond exhibiting an invasive phenotype, the hyperplastic synovium also supports leukocyte recruitment and may be directly activating to the immune system (7,8). Why pannus develops in RA but not in other arthritides also is unknown, but pannus development is central to the progression of joint destruction. Another important clinical question is whether the currently available therapies are effective for treating rheumatoid pannus or whether, as in systemic sclerosis, activated connective tissue is intrinsically resistant to immunosuppression and should be targeted pharmacologically in order to achieve effective disease control. In this issue of Arthritis & Rheumatism, Galligan and Fish present experimental evidence for the participation of circulating fibrocytes in the pathogenesis of inflammatory synovitis (9). The fibrocyte is a monocytederived cell that expresses features of both a macrophage and a connective tissue cell. First identified in the 1990s by their coexpression of hematopoietic progenitor markers together with extracellular matrix proteins, fibrocytes are among the earliest responding cells in the innate response to injury or tissue invasion (10). Fibrocytes normally comprise a minor population of circulating leukocytes, but they are mobilized from the bone marrow and circulate in increased numbers during the systemic inflammatory response, in the setting of ongoing fibrogenesis, or as a consequence of aging (11). Galligan and Fish approached their investigation Richard Bucala, MD, PhD: Yale University School of Medicine, New Haven, Connecticut. Dr. Bucala has received honoraria for service on the Scientific Advisory Board of Promedior (less than $10,000) and owns stock or stock options in the company. Promedior is developing recombinant pentraxin to inhibit fibrocytes. Address correspondence to Richard Bucala, MD, PhD, Department of Medicine, Section of Rheumatology, The Anlyan Center, S521, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520. E-mail: Richard.Bucala@Yale.edu. Submitted for publication June 3, 2012; accepted in revised form June 12, 2012. Arthritis & Rheumatism

I-1 MIF, MIF Alleles, and the Regulation of the Host Response

Abstract: Macrophage migration inhibitory factor (MIF) is the first cytokine activity to be discovered, although it resisted cloning and molecular characterization until relatively late in the era of cytokine discovery. Beyond its eponymous effect on macrophage mobility, MIF now is understood to be a critical upstream regulator of innate immunity that sustains activation responses by mechanisms that include counter-regulating the immunosuppressive action of glucocorticoids and inhibiting stimulus-induced apoptosis. These properties act physiologically to regulate the set-point and the magnitude of an immune response. The recent description of prevalent and functional alleles for MIF and their association with autoimmunity, infection, and cancer has focused attention not only on MIF’s regulatory role in the host response but also on the importance of innate immune pathways in the clinical expression of disease. MIF alleles show significant population stratification, which may reflect the influence of selective pressure, and they likely provide an essential level of variation in innate responsiveness within the human population. Highly homologous orthologues of MIF also have been described in parasitic organisms, and early data suggest that they regulate the host-parasite interaction. First insight into the MIF receptor complex and the structural basis for MIF signal transduction has revealed unique features that hold promise for the pharmacologic modulation of MIF-dependent pathways. Recent developments in MIF biology are reviewed herein and integrated within the concept that MIF allele specific responses influence disease development, whether of an autoimmune, infectious or oncogenic etiology.