Bone Marrow-derived Leukocytes Research Articles

MCT-Dependent Cryptosporidium parvum-Induced Bovine Monocyte Extracellular Traps (METs) under Physioxia.

The apicomplexan protozoan parasite Cryptosporidium parvum is responsible for cryptosporidiosis, which is a zoonotic intestinal illness that affects newborn cattle, wild animals, and people all over the world. Mammalian monocytes are bone marrow-derived myeloid leukocytes with important defense effector functions in early host innate immunity due to their ATP purinergic-, CD14- and CD16-receptors, adhesion, migration and phagocytosis capacities, inflammatory, and anti-parasitic properties. The formation of monocyte extracellular traps (METs) has recently been reported as an additional effector mechanism against apicomplexan parasites. Nonetheless, nothing is known in the literature on METs extrusion neither towards C. parvum-oocysts nor sporozoites. Herein, ATP purinergic receptor P2X1, glycolysis, Notch signaling, and lactate monocarboxylate transporters (MCT) were investigated in C. parvum-exposed bovine monocytes under intestinal physioxia (5% O2) and hyperoxia (21% O2; most commonly used hyperoxic laboratory conditions). C. parvum-triggered suicidal METs were confirmed by complete rupture of exposed monocytes, co-localization of extracellular DNA with myeloperoxidase (MPO) and histones (H1-H4) via immunofluorescence- and confocal microscopy analyses. C. parvum-induced suicidal METs resulted not only in oocyst entrapment but also in hindered sporozoite mobility from oocysts according to scanning electron microscopy (SEM) analyses. Early parasite-induced bovine monocyte activation, accompanied by membrane protrusions toward C. parvum-oocysts/sporozoites, was unveiled using live cell 3D-holotomographic microscopy analysis. The administration of NF449, an inhibitor of the ATP purinergic receptor P2X1, to monocytes subjected to varying oxygen concentrations did not yield a noteworthy decrease in C. parvum-induced METosis. This suggests that the cell death process is not dependent on P2X1. Additionally, blockage of glycolysis in monocyte through 2-deoxy glucose (2-DG) inhibition reduced C. parvum-induced METosis but not significantly. According to monocyte energetic state measurements, C. parvum-exposed cells neither increased extracellular acidification rates (ECAR) nor oxygen consumption rates (OCR). Lactate monocarboxylate transporters (MCT) inhibitor (i.e., AR-C 141990) treatments significantly diminished C. parvum-mediated METs extrusion under physioxic (5% O2) condition. Similarly, treatment with either DAPT or compound E, two selective Notch inhibitors, exhibited no significant suppressive effects on bovine MET production. Overall, for the first time, we demonstrate C. parvum-mediated METosis as P2X1-independent but as an MCT-dependent defense mechanism under intestinal physioxia (5% CO2) conditions. METs findings suggest anti-cryptosporidial effects through parasite entrapment and inhibition of sporozoite excystation.

Potent CCR3 Receptor Antagonist, SB328437, Suppresses Colonic Eosinophil Chemotaxis and Inflammation in the Winnie Murine Model of Spontaneous Chronic Colitis.

Eosinophils and their regulatory molecules have been associated with chronic intestinal inflammation and gastrointestinal dysfunctions; eosinophil accumulation in the gut is prominent in inflammatory bowel disease (IBD). The chemokine receptor CCR3 plays a pivotal role in local and systemic recruitment and activation of eosinophils. In this study, we targeted CCR3-ligand interactions with a potent CCR3 receptor antagonist, SB328437, to alleviate eosinophil-associated immunological responses in the Winnie model of spontaneous chronic colitis. Winnie and C57BL/6 mice were treated with SB328437 or vehicle. Clinical and histopathological parameters of chronic colitis were assessed. Flow cytometry was performed to discern changes in colonic, splenic, circulatory, and bone marrow-derived leukocytes. Changes to the serum levels of eosinophil-associated chemokines and cytokines were measured using BioPlex. Inhibition of CCR3 receptors with SB328437 attenuated disease activity and gross morphological damage to the inflamed intestines and reduced eosinophils and their regulatory molecules in the inflamed colon and circulation. SB328437 had no effect on eosinophils and their progenitor cells in the spleen and bone marrow. This study demonstrates that targeting eosinophils via the CCR3 axis has anti-inflammatory effects in the inflamed intestine, and also contributes to understanding the role of eosinophils as potential end-point targets for IBD treatment.

Myeloid cell‐specific β2AR deletion enhances cardiac hypertrophy and alters leukocyte behavior following pressure overload

As part of the sympathetic nervous system, immune cell‐expressed β2 adrenergic receptor (β2AR) plays an important role in regulating their responses to cardiac injury. We previously demonstrated that deletion of β2AR in all cells of hematopoietic origin resulted in decreased immune cell recruitment, cardiac repair and survival following acute myocardial infarction, however the impact of myeloid cell‐specific β2AR on chronic heart failure (HF)‐induced remodeling remain unknown. Thus, we examined the impact of myeloid cell‐specific β2AR deletion (Lyz2‐Cre x Adrb2fl/fl (LB2) versus Adrb2fl/fl (b2fl) mice) on transaortic constriction (TAC)‐induced HF. Despite a similar initial decline in ejection fraction (EF) at 4 weeks post‐TAC, LB2 mice displayed a better‐preserved EF versus b2fl mice at 8 weeks post‐TAC, which reached a comparable level by 16 weeks post‐TAC. The temporary improvement of cardiac function in the early stage following TAC may be related to an enhanced compensatory hypertrophy in LB2 mice. Indeed, left ventricle posterior wall thickness was higher in LB2 mice especially in the early stage after TAC (4‐8 weeks). This association with enhanced hypertrophy was also observed in an independent model of angiotensin II‐infused LB2 versus b2fl mice with higher expression of Nppa and Nppb observed in LB2 hearts. Via flow cytometry, we observed lower levels of total bone marrow‐derived leukocytes, inflammatory monocytes, macrophages and neutrophils in the hearts of LB2 versus b2fl mice at 16 weeks post‐TAC, whereas total myeloid cells seemed similar between the genotypes. Notably, among the macrophages present in the heart, there was a higher percentage of anti‐inflammatory CD206+ (reparative) macrophages in LB2 mice. In conclusion, these results show that myeloid cell‐specific β2AR regulates cardiac remodeling during the development of chronic HF, which may occur via modulation of the expansion and phenotypic modulation of distinct myeloid cell populations.

P2X7 receptor inhibition ameliorates dendritic spine pathology and social behavioral deficits in Rett syndrome mice

Dysregulated immunity has been implicated in the pathogenesis of neurodevelopmental disorders but its contribution to synaptic and behavioral deficits in Rett syndrome (RTT) remains unknown. P2X7 receptors (P2X7Rs) are unique purinergic receptors with pro-inflammatory functions. Here, we report in a MECP2-deficient mouse model of RTT that the border of the cerebral cortex exhibits increased number of inflammatory myeloid cells expressing cell-surface P2X7Rs. Total knockout of P2X7Rs in MECP2 deficient mice decreases the number of inflammatory myeloid cells, restores cortical dendritic spine dynamics, and improves the animals’ neurological function and social behavior. Furthermore, either genetic depletion of P2X7Rs in bone-marrow derived leukocytes or pharmacological block of P2X7Rs primarily outside of the central nervous system parenchyma, recapitulates the beneficial effects of total P2X7R depletion on the social behavior. Together, our results highlight the pathophysiological roles of P2X7Rs in a mouse model of RTT.

Inflammatory Mediators and Renal Fibrosis.

Renal inflammation is the initial, healthy response to renal injury. However, prolonged inflammation promotes the fibrosis process, which leads to chronic pathology and eventually end-stage kidney disease. There are two major sources of inflammatory cells: first, bone marrow-derived leukocytes that include neutrophils, macrophages, fibrocytes and mast cells, and second, locally activated kidney cells such as mesangial cells, podocytes, tubular epithelial cells, endothelial cells and fibroblasts. These activated cells produce many profibrotic cytokines and growth factors that cause accumulation and activation of myofibroblasts, and enhance the production of the extracellular matrix. In particular, activated macrophages are key mediators that drive acute inflammation into chronic kidney disease. They produce large amounts of profibrotic factors and modify the microenvironment via a paracrine effect, and they also transdifferentiate to myofibroblasts directly, although the origin of myofibroblasts in the fibrosing kidney remains controversial. Collectively, understanding inflammatory cell functions and mechanisms during renal fibrosis is paramount to improving diagnosis and treatment of chronic kidney disease.

In-Depth Proteomic Characterization of Classical and Non-Classical Monocyte Subsets.

Monocytes are bone marrow-derived leukocytes that are part of the innate immune system. Monocytes are divided into three subsets: classical, intermediate and non-classical, which can be differentiated by their expression of some surface antigens, mainly CD14 and CD16. These cells are key players in the inflammation process underlying the mechanism of many diseases. Thus, the molecular characterization of these cells may provide very useful information for understanding their biology in health and disease. We performed a multicentric proteomic study with pure classical and non-classical populations derived from 12 healthy donors. The robust workflow used provided reproducible results among the five participating laboratories. Over 5000 proteins were identified, and about half of them were quantified using a spectral counting approach. The results represent the protein abundance catalogue of pure classical and enriched non-classical blood peripheral monocytes, and could serve as a reference dataset of the healthy population. The functional analysis of the differences between cell subsets supports the consensus roles assigned to human monocytes.

R1R2 peptide ameliorates pulmonary fibrosis in mice through fibrocyte migration and differentiation

Circulating fibrocytes play a key role in the pathogenesis of pulmonary fibrosis. Fibrocytes are bone marrow-derived leukocytes, which enter the lungs in response to their chemoattractant CXCL12 and differentiate into fibroblasts or myofibroblasts, leading to excess deposition of the collagen-rich extracellular matrix. Matrix metalloproteinase (MMP)-9 and MMP-2, secreted by fibrocytes, degrade the subendothelial basement membrane and promote fibrocyte influx into the lungs. Here, we demonstrate that R1R2, a novel peptide derived from the bacterial adhesin SFS, attenuates pulmonary fibrosis by preventing the differentiation of fibrocytes into myofibroblasts and by reducing the invasion of fibrocytes through basement membrane-like proteins. Moreover, our findings reveal dual regulation of R1R2 on MMP-9 through reduced enzymatic activity on gelatin and increased cleavage of CXCL12. These data suggest that R1R2 has potent anti-fibrotic effects against pulmonary fibrosis.

Lp-PLA2 Antagonizes Left Ventricular Healing After Myocardial Infarction by Impairing the Appearance of Reparative Macrophages.

Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6C(high) and reparative Ly-6C(low) monocytes/macrophages. Excessive inflammation disrupts the balance between the 2 phases, impairs infarct healing, and contributes to left ventricle remodeling and heart failure. Lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of the phospholipase A2 family of enzymes, produced predominantly by leukocytes, participates in host defenses and disease. Elevated Lp-PLA2 levels associate with increased risk of cardiovascular events across diverse patient populations, but the mechanisms by which the enzyme elicits its effects remain unclear. This study tested the role of Lp-PLA2 in healing after MI. In response to MI, Lp-PLA2 levels markedly increased in the circulation. To test the functional importance of Lp-PLA2, we generated chimeric mice whose bone marrow-derived leukocytes were Lp-PLA2-deficient (bmLp-PLA2 (-/-)). Compared with wild-type controls, bmLp-PLA2 (-/-) mice subjected to MI had lower serum levels of inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and decreased number of circulating inflammatory myeloid cells. Accordingly, bmLp-PLA2 (-/-) mice developed smaller and less inflamed infarcts with reduced numbers of infiltrating neutrophils and inflammatory Ly-6C(high) monocytes. During the later, reparative phase, infarcts of bmLp-PLA2 (-/-) mice contained Ly-6C(low) macrophages with a skewed M2-prone gene expression signature, increased collagen deposition, fewer inflammatory cells, and improved indices of angiogenesis. Consequently, the hearts of bmLp-PLA2 (-/-) mice healed more efficiently, as determined by improved left ventricle remodeling and ejection fraction. Lp-PLA2 augments the inflammatory response after MI and antagonizes healing by disrupting the balance between inflammation and repair, providing a rationale for focused study of ventricular function and heart failure after targeting this enzyme acutely in MI.

Leukocytes: The Double-Edged Sword in Fibrosis.

Skin tissue scar formation and fibrosis are often characterized by the increased production and deposition of extracellular matrix components, accompanied by the accumulation of a vast number of myofibroblasts. Scaring is strongly associated with inflammation and wound healing to regain tissue integrity in response to skin tissue injury. However, increased and uncontrolled inflammation, repetitive injury, and individual predisposition might lead to fibrosis, a severe disorder resulting in the formation of dense and stiff tissue that loses the physical properties and physiological functions of normal tissue. Fibrosis is an extremely complicated and multistage process in which bone marrow-derived leukocytes act as both pro- and antifibrotic agents, and therefore, few, if any, effective therapies are available for the most severe and lethal forms of fibrosis. Herein, we discuss the current knowledge on the multidimensional impact of leukocytes on the induction of fibrosis, focusing on skin fibrosis.

Invited Commentary

Cardiopulmonary bypass, as developed over the last 50 years, is an extremely effective technique allowing correction of many cardiac lesions. Despite its wide use, many questions still surround its safety and potential risks. Although there have been many improvements in technique and equipment, the inflammatory process initiated with circulatory support remains surprisingly common and surprisingly incompletely understood. This inflammatory process can be system wide or most pronounced in one organ. Pulmonary dysfunction after cardiopulmonary bypass can be particularly problematic. Occasionally, pulmonary injury and inflammatory response can be severe and life threatening. Response of the lungs to cardiopulmonary bypass has been studied clinically and experimentally since the technique's introduction. From these studies, we have come to understand that the initiating insult is most often cardiogenic, infectious, or inflammatory in origin. The inflammatory response is theorized to arise from contact of blood with the artificial material of the bypass circuit, leading to complement activation, neutrophil and leukocyte activation, and activation of various enzyme systems. Molecular biologists have made a plethora of advances in understanding the inflammatory response. Thoracic surgeons have been slow to apply these advances to our own field in general and to pulmonary dysfunction in particular. In this issue of The Annals, Goto and colleagues [1Goto Y. Hiramatsu Y. Ageyama N. et al.Cardiopulmonary bypass induces recruitment of bone marrow-derived leukocytes to the lungs in monkeys.Ann Thorac Surg. 2014; 97: 617-623Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar] present an experimental study concerning the inflammatory response mounted in response to cardiopulmonary bypass. Using an elegant technique that they developed in a primate model, they document a rapid release of both polymorphonuclear leukocytes (PMN) and monocytes (MO) from the bone marrow into both the circulation and bronchoalveolar lavage fluid after cardiopulmonary bypass. A thymidine analog, 5'-bromo-2'-deoxyuridine (BrdU), was used to label dividing leucocytes in the bone marrow. Flow cytometry was used to document the increased accumulation of BrdU-positive polymorphonuclear neutrophils and monocytes in the peripheral blood and in the bronchoalveolar lavage fluid in both experimental and control animals. The PMNs and MOs first appear in the peripheral blood at 24 hours and peak at 120 hours after bypass. In contrast to the peripheral blood, bronchoalveolar fluid washings from experimental animals contain significantly more BrdU-labeled cells (PMNs and MOs) within 24 hours than do control animals. This type of elegant primate study has somewhat fallen out of favor owing to regulatory and procedural difficulties. Goto and colleagues [1Goto Y. Hiramatsu Y. Ageyama N. et al.Cardiopulmonary bypass induces recruitment of bone marrow-derived leukocytes to the lungs in monkeys.Ann Thorac Surg. 2014; 97: 617-623Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar] are to be commended for their important and challenging work using a classic animal experimental design that includes a control group. This type of study is extremely important in improving our understanding of cardiopulmonary bypass. This improved understanding of the inflammatory reaction after cardiopulmonary bypass opens the door to therapeutic interventions at the molecular level that might limit and ameliorate pulmonary dysfunction after cardiac surgery. Cardiopulmonary Bypass Induces Recruitment of Bone Marrow–Derived Leukocytes to the Lungs in MonkeysThe Annals of Thoracic SurgeryVol. 97Issue 2PreviewA bone marrow (BM) response induced by cardiopulmonary bypass (CPB) as a systemic inflammatory reaction has previously been postulated but not clarified. Newly released polymorphonuclear leukocytes (PMNs) and monocytes from the BM are known to be immature, indicating their greater potential to damage tissue. The present study aimed to examine the kinetics of BM–derived leukocytes associated with CPB in a nonhuman primate model. Full-Text PDF

White blood count and infarct size, myocardial salvage and clinical outcomes: the role of differentials

Evidence suggests that inflammation, both local and systemic, plays an important role in the pathogenesis of acute myocardial infarction [1]. There is also evidence that increased concentrations of inflammatory markers such as C-reactive protein interleukin-2 and tumor necrosis factor can identify patients at high risk of future ischemic events, suggesting that the intensity of the inflammatory response influences clinical outcome in acute coronary syndromes [2, 3]. Multiple studies have demonstrated also an association between elevated leucocyte count, which is a non-specific marker of inflammation, and the extent of coronary artery disease or increased short-term or long-term risk of death in patients with various manifestations of coronary artery disease [4, 5]. Other studies [6] have shown that total leucocyte count, but not C-reactive protein, predicts 1-year mortality in patients with acute coronary syndromes treated with percutaneous coronary intervention. Furthermore, recent studies have emphasized the usefulness of neutrophil to lymphocyte ratio in predicting short-and long-term mortality, thrombus formation, infarct related artery patency, TIMI flow grade after primary coronary angioplasty, impaired myocardial perfusion, ventricular arrhythmias and critical limb ischemia [7–9]. Indeed, in patients with non-ST-elevation myocardial infarction a ratio above 4.7 has been reported to be an independent predictor of short-term and long-term mortality [7] and ratio[3.3 was found to be an independent predictor of impaired coronary flow after primary percutaneous coronary intervention and of in-hospital major adverse cardiac events in patients with ST-segment elevation myocardial infarction [10]. In a recent issue of the Int J Cardiovasc Imaging [11] and additional very interesting paper was published which shows that in patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction an elevated baseline white blood count is associated with less salvaged myocardium, larger infarct size and poorer clinical outcomes. All these papers and results really suggest a key role of inflammation in coronary artery disease. However, differential count was not available in these studies, and therefore, assessing the relative impact of white blood count subpopulations on myocardial infarction was not reported. Total white blood count comprises several cell types such as monocytes, lymphocytes and eosinophils all of which are implicated in the development, progression and instability of atherosclerotic plaque. None of the above studies have focused attention on the presence of eosinophils and this was probably based on an older study which has suggested that total white blood count is a better correlate of long-term than differentials [12]. Eosinophils, however, play an important role in inflammation and can identify the etiology of inflammation since they denote hypersensitivity inflammation [13–15]. Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of inflammatory responses and thus have important roles in the pathogenesis of inflammatory diseases. These cells are also bone marrow-derived granulocytic leukocytes which express H4 histamine receptors in their surface. These receptors facilitate eosinophil chemotaxis toward mast cells which are the major producers of an array of inflammatory soluble mediators. These inflammatory mediators can induce the coronary hypersensitivity associated Kounis syndrome manifesting as vasospastic angina and/or acute myocardial N. G. Kounis (&) Medical Sciences, Patras Highest Institute of Education and Technology, Patras, Achaia, Greece e-mail: ngkounis@otenet.gr

Eosinophilic coronary periarteritis presenting with vasospastic angina and sudden death: a new cause and manifestation of Kounis syndrome?

Dear Editor, In a very interesting report published in Virchows Archives [1], seven patients were described suffering from angina pectoris who died suddenly in the morning hours. Histological diagnosis revealed a new pathological entity, which was named eosinophilic coronary periarteritis. All of these patients had inflammatory infiltrates, mainly eosinophilic, in coronary adventitia and periadventitial soft tissue. One of them also had eosinophilic infiltration of the intima and another had infiltration of the medial smooth muscle layers by a small number of eosinophils. This report raises important issues concerning the causality and clinical presentation of hypersensitivity-associated acute coronary syndrome, also known as Kounis syndrome [2]. This is a hypersensitivity angina syndrome, which can progress to acute myocardial infarction and sudden death. It is a ubiquitous disease affecting patients of any age, with a continuously increasing list of causes, broadening clinical manifestations, and covering a wide spectrum of activation disorders of mast cells and other interrelated and interacting inflammatory cells. Platelets with FcγRI, FcγRII, FceRI, and FceRII receptors are also involved in the development of this syndrome. Kounis syndrome can complicate the Churg–Strauss syndrome, which is sometimes associated with coronary artery spasm with repeated angina pectorislike attacks and/or acute myocardial infarction [3]. Eosinophilic coronary arteritis can also complicate Churg–Strauss syndrome and can clinically present as type I and type II variants of Kounis syndrome [2]. Eosinophilic coronary periarteritis might be a further evolution of entities previously described as “isolated eosinophilic coronary arteritis,” “eosinophilic arteritis,” “eosinophilic coronary arteritis,” and “limited form of Churg–Strauss syndrome.” Indeed, the first description already more than six decades ago was named “diffuse eosinophilic arteritis” [4]. Although the authors of the above report [1] did not describe any presence of mast cells during histological examination, other reports emphasized eosinophilic infiltration of adventitia, media, and intimal subendothelium together with diffusely distributed mast cells [5]. This could be an important finding because eosinophilic receptors facilitate eosinophil chemotaxis toward mast cells that are the major producers of an array of inflammatory soluble mediators, including histamine, which can induce Kounis syndrome. Soluble mediators secreted by mast cells and eosinophils modulate reciprocal interactions between these two cells in the socalled “allergic effector unit.” Eosinophils are bone marrow-derived granulocytic leukocytes, which normally reside in tissues and express H4 histamine receptors. Eosinophils respond to histamine in changing cell shape and upregulation of adhesion molecules and chemotaxis, and these responses are mediated by the H4 receptor N. G. Kounis (*) Department of Medical Sciences, Patras Highest Institute of Education and Technology, Queen Olgas Square, 7 Aratou Street, Patras 26221, Greece e-mail: ngkounis@otenet.gr

Mast cells are present in the choroid of the normal eye in most vertebrate classes

Mast cells are bone marrow-derived tissue-homing leukocytes, which have traditionally been regarded as effector cells in allergic disorders, responses against parasites, and regulation of blood flow, but a broader perspective of their functional heterogeneity, such as immunomodulation, angiogenesis, tissue repair, and remodeling after injury, is now emerging. The persistence of mast cells in connective tissues throughout the evolution of vertebrates is evidence of strong selective pressure suggesting that these cells must have multiple beneficial and important roles in normal homeostasis. While mast cells are present within the uveal tract of eutherian mammals, there is little known about their presence in the choroid of other vertebrate classes. Eye tissues from a range of vertebrate species (fish, amphibian, reptiles, birds, marsupials, monotreme, and eutherian mammals) were investigated. Tissues were fixed in either 2% glutaraldehyde, 2% paraformaldehyde or a mixture of both and processed for resin embedding. Semi-thin sections of the retina and choroid were cut and stained with toluidine blue. Mast cells were identified in the choroid of all classes of vertebrates investigated except sharks. Their morphology, location, and staining characteristics were remarkably similar from teleost fish through to eutherian mammals and bore close morphological resemblance to mammalian connective tissue mast cells. The similar morphology and distribution of mast cells in the choroid of all vertebrate classes studied suggest a basic physiological function that has been retained since the evolution of the vertebrate eye.

CD11b+ Bone Marrow–Derived Monocytes Are the Major Leukocyte Subset Responsible for Retinal Capillary Leukostasis in Experimental Diabetes in Mouse and Express High Levels of CCR5 in the Circulation

We investigated the phenotype of cells involved in leukostasis in the early stages of streptozotocin-induced diabetes in mice by direct observation and by adoptive transfer of calcein-AM-labeled bone marrow-derived leukocytes from syngeneic mice. Retinal whole mounts, confocal microscopy, and flow cytometry ex vivo and scanning laser ophthalmoscopy in vivo were used. Leukostasis in vivo and ex vivo in retinal capillaries was increased after 2 weeks of diabetes (Hb A(1c), 14.2 ± 1.2) when either donor or recipient mice were diabetic. Maximum leukostasis occurred when both donor and recipient were diabetic. CD11b(+), but not Gr1(+), cells were preferentially entrapped in retinal vessels (fivefold increase compared with nondiabetic mice). In diabetic mice, circulating CD11b(+) cells expressed high levels of CCR5 (P = 0.04), whereas spleen (P = 0.0001) and retinal (P = 0.05) cells expressed increased levels of the fractalkine chemokine receptor. Rosuvastatin treatment prevented leukostasis when both recipient and donor were treated but not when donor mice only were treated. This effect was blocked by treatment with mevalonate. We conclude that leukostasis in early diabetic retinopathy involves activated CCR5(+)CD11b(+) myeloid cells (presumed monocytes). However, leukostasis also requires diabetes-induced changes in the endothelium, because statin therapy prevented leukostasis only when recipient mice were treated. The up-regulation of the HMG-CoA reductase pathway in the endothelium is the major metabolic dysregulation promoting leukostasis.

Prostaglandin E2 in Remote Control of Myocardial Remodeling

Prostaglandin E2 in Remote Control of Myocardial Remodeling

Conditions of Retinal Glial and Inflammatory Cell Activation after Irradiation in a GFP-Chimeric Mouse Model

Microglia cells have been associated with immunologic defense and repair. The course of retinal disease after lethal irradiation for bone marrow depletion and substitution was evaluated with respect to macrophage and microglial involvement. Lethal irradiation in C57BL/6 mice was conducted with a low-voltage radiation unit. The animals were randomized to shielded or unshielded radiation and subsequently received transplants of GFP+ bone marrow cells (beta-actin promoter). The GFP transformation rate was analyzed by flow cytometry. GFP+ cells in the retina were examined for co-localization with macrophage and dendritic cell markers at various time points between 1 and 7 months after irradiation. Clodronate liposomes were used to investigate the fate of migrated and residential microglia cells. Pathologic angiogenesis was investigated in laser-induced choroidal neovascularization (CNV) after unshielded and shielded irradiation. Flow cytometry revealed average transformation rates of 78.2% in unshielded and 64.1% in shielded group. Four weeks after transplantation, perfused flat mounts were virtually free of extravasal GFP+ cells in both groups, whereas 4 months after irradiation, cluster cell infiltrations, preferentially in the peripheral retina, became apparent exclusively in the unshielded group. Cell morphology ranged from oval, to a few extensions, to dendritiform with long-branched extensions. Clodronate treatment resulted in a reduction of GFP+ cells in the retinal tissue when applied 3 months after unshielded irradiation. Although GFP+ cells accumulated in the choroidal scar after laser treatment, in both the shielded and unshielded groups, GFP+ cells in the overlying retina were restricted to the unshielded group. Approximately 3 months after lethal full-body irradiation including the eye, bone marrow-derived leukocytes exhibit a wound-healing reaction, and unlike physiological turnover, infiltrate the retina and form microglial cells.

The K1 Serotype Capsular Polysaccharide ofPorphyromonas gingivalisElicits Chemokine Production from Murine Macrophages That Facilitates Cell Migration

Porphyromonas gingivalis is the principal organism associated with aggressive forms of generalized periodontal disease. Previous reports have suggested that encapsulated P. gingivalis strains are more virulent than unencapsulated strains; however, the contribution of capsular polysaccharide (CPS) to the virulence of this organism is poorly understood. Since periodontal disease presents with a complex inflammatory cell lesion comprised of neutrophils and monocytes, we cultured murine peritoneal macrophages with heat-killed P. gingivalis W83, CPS purified from P. gingivalis strain W83, and the seven known serotype-specific P. gingivalis CPS and assessed the ability of supernatant fluids produced by challenged macrophages to attract naïve inflammatory cells. We also defined JE/MCP-1, KC, MIP-2, and RANTES production in response to the P. gingivalis CPS antigens. We observed that supernatant fluids collected from macrophages incubated with P. gingivalis W83 and serotype K1 CPS stimulated the migration of naïve murine bone marrow-derived polymorphonuclear leukocytes in an in vitro cell migration chamber. CPS from W83 and the K1 serotype elicited potent chemokine secretion patterns for macrophages, while those specific to serotypes K2 to K7 were significantly less stimulatory. Reverse transcription-PCR and enzyme-linked immunosorbent assay revealed JE/MCP-1, KC, MIP-2, and RANTES expression from murine macrophages which had been challenged with purified P. gingivalis W83 CPS. Chemokine production appeared to be dependent on both the dose of and time of exposure to P. gingivalis W83 CPS. These data demonstrate that the P. gingivalis serotype K1 CPS elicits chemokine production from phagocytic cells. Furthermore, these data suggest that the host response to this antigen may contribute to the formation of the inflammatory cell lesion observed during P. gingivalis-elicited periodontal disease.

Attenuated pseudorabies virus-evoked rapid innate immune response in the rat brain

Ba-DupGreen (BDG) is a highly attenuated, Bartha-derived pseudorabies virus (PRV) expressing green fluorescent protein (GFP) with immediate-early kinetics. Innate immune mechanisms underlying the low infectivity of the virus and the disappearance of infected neurons from the brain were studied at cellular level following injection of BDG into the spleen. The temporal shift in the expression between GFP and viral structural proteins allowed us to discriminate three stages of viral infection in the compromised neurons in correlation with the ongoing local inflammatory response. Iba1/lectin/OX42-positive microglia were recruited to infected neurons within 4–6 h following the initiation of virus replication, incorporated BrdU, isolated the infected cells before the disintegration of their membranes and phagocytosed collapsed neurons. Ex vivo-labeled blood and bone marrow-derived leukocytes, including ED-1-positive macrophages were involved in the immune cell assembly around compromised neurons, which resulted in the complete clearance of infected neurons from the early-infected brain regions.

Human Dendritic Cells: Potent Antigen-Presenting Cells at the Crossroads of Innate and Adaptive Immunity

Dendritic cells (DCs) are specialized, bone marrow-derived leukocytes that are critical to the development of immunity. Investigators have emphasized the role of DCs in initiating adaptive or acquired MHC-restricted, Ag-specific T cell responses. More recent evidence supports important roles for DCs in the onset of innate immunity and peripheral tolerance. Progress in the generation of DCs from defined hemopoietic precursors in vitro has revealed the heterogeneity of these APCs and their attendant divisions of labor. This review will address these developments in an attempt to integrate the activities of different DCs in coordinating innate and adaptive immunity.

Reduced Macrophage Apoptosis Is Associated With Accelerated Atherosclerosis in Low-Density Lipoprotein Receptor-Null Mice

The majority of apoptotic cells in atherosclerotic lesions are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains unclear. Elevated expression of Bax, one of the pivotal proapoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol-treated mouse macrophages. In this study, we examined the effect of Bax deficiency in bone marrow-derived leukocytes on the development of atherosclerosis in low-density lipoprotein receptor-null (LDLR-/-) mice. Fourteen 8-week-old male LDLR-/- mice were lethally irradiated and reconstituted with either wild-type (WT) C57BL6 or Bax-null (Bax-/-) bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and Bax-/- group. However, quantitation of cross sections from proximal aorta revealed a 49.2% increase (P=0.0259) in the mean lesion area of the Bax-/- group compared with the WT group. A 53% decrease in apoptotic macrophages in the Bax-/- group was found by TUNEL staining (P<0.05). The reduction of apoptotic activity in macrophages stimulates atherosclerosis in LDLR-/- mice, which is consistent with the hypothesis that macrophage apoptosis suppresses the development of atherosclerosis.