Heterogeneous Population Of Macrophages Research Articles

Autofluorescence imaging of endogenous metabolic cofactors in response to cytokine stimulation of classically activated macrophages.

Macrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates. Here, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation. Based on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population. Overall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation.

Measuring the Metabolic State of Tissue-Resident Macrophages via SCENITH.

Functional reprograming of cells is linked to a process of metabolic rewiring that is adapted for such new functions or microenvironment. Macrophages are present in all tissues and exposed to different microenvironments throughout our body. Profiling energetic metabolism of tissue resident and other heterogeneous populations of macrophages in vitro and ex vivo is technologically very challenging. We have recently developed a method to functionally profile energetic metabolism with single-cell resolution, named SCENITH. This method can be performed rapidly ex vivo and does not require specialized equipment. In this book chapter, we will summarize the tissue processing, the procedure and methods, the analysis and example of results, and a series of frequently asked questions.

Whole-Mount Imaging of Adipose Tissue Macrophages.

The adipose tissue comprises highly heterogeneous macrophage populations, which play critical roles in the regulation of adipose tissue function and dysfunction during health and disease. Whole-amount staining is a powerful technique for macrophage characterization within the 3D environment of the adipose tissue, enabling the visualization of different macrophage populations and their interaction with other cells within their in vivo niche. Due to the high-fat content and softness, freezing and sectioning of adipose tissue is difficult, and distortion of tissue morphology typically occurs, especially in the case of white adipose tissue. We describe here a whole-mount staining alternative for adipose tissue imaging that preserves all structures and allows high-resolution image acquisition. We address in a step-by-step manner how to perform immunofluorescence staining of different fat pads and how to optimally visualize cellular and acellular (extracellular matrix) constituents of the adipose tissue and its vasculature, as well as resident and infiltrating macrophage populations.

Abstract 502: Trem2 Regulates Foamy Macrophage Formation In Atherosclerotic Plaque Formation

Atherosclerosis is driven by the accumulation of lipid in artery walls, leading to plaque formation. Plaque expansion is driven by monocyte infiltration where they differentiate into heterogeneous macrophage populations, including foamy macrophages. To understand genes that regulate monocyte differentiation toward foamy macrophages, we performed pseudotime analysis of murine scRNA-seq from atherosclerotic plaques. This analysis identified Trem2, a lipid sensor expressed on myeloid cells, as a potential regulator of foamy cell formation. These results were further supported by an in vitro genome-wide crispr screen performed on oxLDL-treated foamy macrophages, which identified Trem2 as a candidate regulator for oxLDL uptake. To test the role of macrophage Trem2 in atherosclerotic plaque formation, we generated Trem2 fl/fl CX3CR1 CreER LDLR -/- mice that delete Trem2 only in macrophages upon tamoxifen treatment (TAM). TAM enriched Western Diet (WD) feeding for 8 weeks lead to significantly decreased plaque formation in the aortic arch and aortic sinus of Trem-deficient mice compared to litermate control. Importantly, Trem2flox animals displayed comparable serum cholesterol levels to control mice following WD feeding. Finally, to determine whether Trem2 is required for foamy macrophage formation in vivo, we performed mixed bone marrow chimera of 50% Trem2 -/- marrow and 50% control marrow into Ldlr -/- recipients fed with western diet. Plaque associated Trem2-deficient macrophages failed to develop into foamy macrophages when in competition with WT macrophages, despite normal ratios of circulating monocytes. Overall, this data identifies Trem2 as a novel regulator of foamy macrophage formation and atherosclerotic plaque growth that could be targeted therapeutically to treat CVD.

Macrophages in the heart: Active players or simple bystanders?

Today, more and more studies focus on the processes in which macrophages are involved. These discoveries provide new perspectives on the cellular mechanisms that regulate the physiological functions of the healthy heart. Moreover, they offer a deeper knowledge of the pathologic processes underlying the onset and the evolution of specific cardiac impairment. The heterogeneous population of macrophages within the heart can be divided by origin, expression profile, and function. The pool of cardiac macrophages includes at least two distinct subsets with different ontogeny. The first one has an embryonic origin, deriving from the yolk sac and the fetal liver, while the other macrophage subset results from the postnatal recruitment of monocytes produced in the bone marrow. This review will focus on new phenotypes and functions of cardiac macrophages that have been identified in the last years and that need to be deeply studied to unveil new potential therapies aimed at treating cardiac diseases.

STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

BackgroundIn breast cancer, complex interactions between tumor cells and cells within the surrounding stroma, such as macrophages, are critical for tumor growth, progression, and therapeutic response. Recent studies have highlighted the complex nature and heterogeneous populations of macrophages associated with both tumor-promoting and tumor-inhibiting phenotypes. Defining the pathways that drive macrophage function is important for understanding their complex phenotypes within the tumor microenvironment. Signal transducer and activator of transcription (STAT) transcription factors, such as STAT5, are key regulators of immune cell function. The studies described here investigate the functional contributions of STAT5 to tumor-associated macrophage function in breast cancer.MethodsInitial studies were performed using a panel of human breast cancer and mouse mammary tumor cell lines to determine the ability of tumor cell-derived factors to induce STAT5 activation in macrophages. Further studies used these models to identify soluble factors that activate STAT5 in macrophages. To delineate STAT5-specific contributions to macrophage function, a conditional model of myeloid STAT5 deletion was used for in vitro, RNA-sequencing, and in vivo studies. The effects of STAT5 deletion in macrophages on tumor cell migration and metastasis were evaluated using in vitro co-culture migration assays and an in vivo tumor cell-macrophage co-injection model.ResultsWe demonstrate here that STAT5 is robustly activated in macrophages by tumor cell-derived factors and that GM-CSF is a key cytokine stimulating this pathway. The analysis of RNA-seq studies reveals that STAT5 promotes expression of immune stimulatory genes in macrophages and that loss of STAT5 in macrophages results in increased expression of tissue remodeling factors. Finally, we demonstrate that loss of STAT5 in macrophages promotes tumor cell migration in vitro and mammary tumor metastasis in vivo.ConclusionsBreast cancer cells produce soluble factors, such as GM-CSF, that activate the STAT5 pathway in macrophages and drive expression of inflammatory factors. STAT5 deletion in myeloid cells enhances metastasis, suggesting that STAT5 activation in tumor-associated macrophages protects against tumor progression. Understanding mechanisms that drive macrophage function in the tumor microenvironment will ultimately lead to new approaches that suppress tumor-promoting functions while enhancing their anti-tumor functions.

Heterogeneous macrophages contribute to the pathology of disc herniation induced radiculopathy

Macrophages play important roles in the progression of intervertebral disc herniation and radiculopathy. To better understand the roles of macrophages in this process, we developed a new mouse model that mimics human radiculopathy. A preclinical randomized animal study. Three types of surgeries were performed in randomly assigned Balb/c mice. These were spinal nerve exposure, traditional anterior disc puncture, and lateral disc puncture with nerve exposure (n=16/group). For the nerve exposure group, the left L5 spinal nerve was exposed without disc injury. For the traditional anterior puncture, L5/6 disc was punctured by an anterior approach as previously established. For lateral puncture with nerve exposure, the left L5 spinal nerve was exposed by removing the psoas major muscle fibers, and the L5/6 disc was punctured laterally on the left side with a 30G needle, allowing the nucleus to protrude toward the L5 spinal nerve. Mechanical hyperalgesia (pain sensitivity) of hind paws was assessed with electronic von Frey assay on alternative day for up to 2 weeks. MRI, histology, and immunostaining were performed to confirm disc herniation and inflammation. Ipsilateral pain in the lateral puncture with nerve exposure group was significantly greater than the other groups. Pro-inflammatory cytokines IL-1β and IL-6 were markedly elevated at the hernia sites of both puncture groups and the spinal nerve of lateral puncture with never exposure group on postoperative day 7. Heterogeneous populations of macrophages were detected in the infiltration tissue of this mouse model and in tissue from patients undergone discectomy. We have established a new mouse model that mimics human radiculopathy and demonstrated that a mixed phenotype of macrophages contribute to the pathogenesis of acute discogenic radiculopathy. This study provides a clinically relevant in vivo animal model to elucidate complex interactions of disc herniation and radicular pain, which may present opportunities for the development of macrophage-anchored therapeutics to manage radiculopathy.

Immunophenotypic analysis of the distribution of hepatic macrophages, lymphocytes and hepatic stellate cells in the adult rat liver.

The liver consists of parenchymal hepatocytes and non-parenchymal cells. Non-parenchymal cells, Kupffer cells, hepatic stellate cells and cholangiocytes have crucial roles in liver homeostasis and liver pathology. To establish baseline data, this study investigated immunohistochemically the distribution of non-parenchymal cells in perivenular areas (PV), periportal areas (PP) and Glisson's sheath (GS) of adult rat liver. Liver tissues were collected from the left lateral lobe of rats. CD163-positive macrophages were seen along the sinusoid of PV and PP areas, indicating Kupffer cells. Double immunofluorescence showed, Kupffer cells partly co-expressed CD68 and MHC class II antigens in the liver. The numbers of Kupffer cells were significantly high in PP areas as compared with PV or GS areas. CD68-positive exudative macrophages were highly localized in PP and GS areas and a comparatively low PV area. MHC class II-positive dendritic cells (activated macrophages) were localized mainly in GS. Granzyme B-positive NK cells were mainly localized in the Glisson's sheath. CD3-positive T cells and CD20-positive B cells were distributed along the sinusoids of the PP and PV areas of hepatic lobules. Vimentin and glial fibrillary acidic protein (GFAP)-positive hepatic stellate cells were localized along sinusoids in the hepatic lobules of the liver. Cholangiocytes reacting to cytokeratin 19 were seen on interlobular bile ducts in Glisson's sheath of the liver. This study shows that heterogeneous macrophage populations, liver-resident lymphocytes and hepatic stellate cells localized in PP and PV areas or GS areas of the liver with cells specific patterns.

B cells modulate the expression of MHC-II on cardiac CCR2− macrophages

The uninjured murine heart contains a heterogeneous population of macrophages with disparate ontogenies and functions. These macrophages are often associated with blood vessels and can be subclassified based on the expression of CC chemokine receptor 2 (CCR2) and major histocompatibility complex class II (MHC-II). The biological cues that modulate these macrophage pool subpopulations have not been completely identified. It has been recently shown that a sub-population of circulating naïve B cells adheres to the myocardial microvasculature. We hypothesized that B cells might modulate the phenotype of myocardial macrophages. To test this hypothesis, we analyzed both the relative location of B cells and macrophages in myocardial histological section and the prevalence of myocardial macrophage subsets in hearts from B cell–deficient mice (μMT) and mice depleted of B cells through administration of an anti-CD20 antibody. We found that B cells pause in the microvasculature in proximity of macrophages and modulate the number of myocardial CCR2−MHC-IIhigh cells. Through in vitro studies we found that this is likely the result of a paracrine effect of B cells on the expression of MHC-II in CCR2− cells. These results reveal an unexpected relationship between B cells and resident macrophages and, highlighting a direct intramyocardial effect of circulating B cells, challenge the currently held belief that naïve recirculating B lymphocytes merely shuttle between lymphoid stations.

Role of Macrophages in Cardioprotection.

Cardiovascular diseases are the leading cause of mortality worldwide. It is widely known that non-resolving inflammation results in atherosclerotic conditions, which are responsible for a host of downstream pathologies including thrombosis, myocardial infarction (MI), and neurovascular events. Macrophages, as part of the innate immune response, are among the most important cell types in every stage of atherosclerosis. In this review we discuss the principles governing macrophage function in the healthy and infarcted heart. More specifically, how cardiac macrophages participate in myocardial infarction as well as cardiac repair and remodeling. The intricate balance between phenotypically heterogeneous populations of macrophages in the heart have profound and highly orchestrated effects during different phases of myocardial infarction. In the early “inflammatory” stage of MI, resident cardiac macrophages are replaced by classically activated macrophages derived from the bone marrow and spleen. And while the macrophage population shifts towards an alternatively activated phenotype, the inflammatory response subsides giving way to the “reparative/proliferative” phase. Lastly, we describe the therapeutic potential of cardiac macrophages in the context of cell-mediated cardio-protection. Promising results demonstrate innovative concepts; one employing a subset of yolk sac-derived, cardiac macrophages that have complete restorative capacity in the injured myocardium of neonatal mice, and in another example, post-conditioning of cardiac macrophages with cardiosphere-derived cells significantly improved patient’s post-MI diagnoses.

Macrophage heterogeneity in the context of rheumatoid arthritis

Macrophages are very important in the pathogenesis of rheumatoid arthritis (RA). The increase in the number of sublining macrophages in the synovium is an early hallmark of active rheumatic disease, and high numbers of macrophages are a prominent feature of inflammatory lesions. The degree of synovial macrophage infiltration correlates with the degree of joint erosion, and depletion of these macrophages from inflamed tissue has a profound therapeutic benefit. Research has now uncovered an unexpectedly high level of heterogeneity in macrophage origin and function, and has emphasized the role of environmental factors in their functional specialization. Although the heterogeneous populations of macrophages in RA have not been fully characterized, preliminary results in mouse models of arthritis have contributed to our understanding of the phenotype and ontogeny of synovial macrophages, and to deciphering the properties of monocyte-derived infiltrating and tissue-resident macrophages. Elucidating the molecular mechanisms that drive polarization of macrophages towards proinflammatory or anti-inflammatory phenotypes could lead to identification of signalling pathways that inform future therapeutic strategies.

In Vivo Approaches Reveal a Key Role for DCs in CD4+ T Cell Activation and Parasite Clearance during the Acute Phase of Experimental Blood-Stage Malaria

Dendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.

The involvement of the monocytes/macrophages in chronic inflammation associated with atherosclerosis

Atherosclerosis is a progressive chronic disease of large and medium arteries, characterized by the formation of atherosclerotic plaques. Monocytes and macrophages are key factors in lesion development, participating to the processes that mediate the progression of the atherosclerotic plaque (lipid accumulation, secretion of pro-inflammatory and cytotoxic factors, extracellular matrix remodeling). The recruitment of the monocytes in the vascular wall represents a hallmark in the pathology of the atherosclerotic lesion. Monocyte adhesion and transmigration are dependent on the complementary adhesion molecules expressed on the endothelial surface, whose expression is modulated by chemical mediators. The atherosclerotic plaque is characterized by a heterogeneous population of macrophages reflecting the complexity and diversity of the micro-environment to which cells are exposed after entering the arterial wall. Within the atherosclerotic lesions, macrophages differentiate, proliferate and undergo apoptosis. Taking into account that their behavior has a direct and critical influence on all lesional stages, the development of therapeutic approaches to target monocytes/macrophages in the atherosclerotic plaque became a focal interest point for researchers in the field.

Resident Alveolar Macrophages Are Susceptible to and Permissive of Coxiella burnetii Infection

Coxiella burnetii, the causative agent of Q fever, is a zoonotic disease with potentially life-threatening complications in humans. Inhalation of low doses of Coxiella bacteria can result in infection of the host alveolar macrophage (AM). However, it is not known whether a subset of AMs within the heterogeneous population of macrophages in the infected lung is particularly susceptible to infection. We have found that lower doses of both phase I and phase II Nine Mile C. burnetii multiply and are less readily cleared from the lungs of mice compared to higher infectious doses. We have additionally identified AM resident within the lung prior to and shortly following infection, opposed to newly recruited monocytes entering the lung during infection, as being most susceptible to infection. These resident cells remain infected up to twelve days after the onset of infection, serving as a permissive niche for the maintenance of bacterial infection. A subset of infected resident AMs undergo a distinguishing phenotypic change during the progression of infection exhibiting an increase in surface integrin CD11b expression and continued expression of the surface integrin CD11c. The low rate of phase I and II Nine Mile C. burnetii growth in murine lungs may be a direct result of the limited size of the susceptible resident AM cell population.

Macrophage markers and C3d in the central & peripheral choroid of young, aged and amd eyes

AbstractPurpose To investigate the distribution and expression of macrophage phenotypes and the complement activation protein, C3d, in human choroid/retina in young, aged and AMD eyes.Methods Paraffin sections of central and peripheral choroid/retina from young (<40 years, n=6), aged (>70 years, n=4) and AMD (>70 years, n=8) human post‐mortem eyes were co‐immunolabelled with antibodies to leukocyte and macrophage markers (CD68, CD163, Iba1 & CD45) and complement activation component, C3d. Localisation and distribution of antibodies was assessed using confocal microscopy. Counts of macrophage sub‐populations were compared in central and peripheral choroid for each group.Results Heterogeneous populations of macrophages were found in the choroid/retina of all groups. Iba1+ and CD45+ retinal microglia and macrophages, and choroidal macrophages were observed in all eyes. In AMD eyes, Iba1+ and CD45+ cells were seen in the subretinal space amongst photoreceptors, as well as in inner retina. Compared to young eyes, aged and AMD specimens showed increased numbers of CD163+ macrophages (M2, proangiogenic) in choroid, and in central vs peripheral retina. Fewer CD68+ cells were seen in all specimens, regardless of age or location. C3d was expressed in young, aged and AMD eyes, localised to Bruch’s membrane and choriocapillaris, with more extensive immunostaining in aged and AMD eyes. C3d was also seen in drusen in aged and AMD eyes, and more peripherally, compared to young eyes.Conclusion Age‐related changes in macrophage phenotypes and in complement pathway activation at the choroidal/retinal interface may play a role in development and progression of AMD. Supported by Sydney Foundation for Medical Research.

α1-Antitrypsin Production by Proinflammatory and Antiinflammatory Macrophages and Dendritic Cells

α(1)-Antitrypsin (AAT) acts as an important neutrophil elastase inhibitor in the lung. Although the hepatocyte is considered to be the primary source of AAT, local production by monocytes, macrophages, and epithelial cells may contribute to the formation of an antielastase screen. Because monocytes can differentiate into a heterogeneous population of macrophages with subpopulations ranging from proinflammatory properties (MΦ-1) to antiinflammatory properties (ΜΦ-2) and into dendritic cells (DCs), we studied whether LPS, TNF-α, and oncostatin M (OSM) enhance AAT production differentially in cultured ΜΦ-1, ΜΦ-2, and DCs. Monocytes from healthy blood donors were cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor, or GM-CSF with IL-4 to obtain ΜΦ-1, ΜΦ-2, and immature (i)DCs, respectively. Cells were stimulated with LPS, TNF-α, or OSM, and AAT synthesis was assessed by quantitative RT-PCR, immunocytochemistry, and ELISA. Spontaneous release of AAT was higher in ΜΦ-1 than in ΜΦ-2 and iDCs, and only LPS significantly increased AAT production in ΜΦ-1, ΜΦ-2, and DC. TNF-α and OSM did not affect AAT secretion. The secretion levels of the related protease inhibitors α-1 antichymotrypsin and secretory leukocyte proteinase inhibitor were below the limits of detection by ELISA. In contrast to the protein data, analysis by quantitative RT-PCR showed that 24-hour LPS exposure caused a maximal 2.1-fold AAT mRNA increase in ΜΦ-1, a 21-fold increase in ΜΦ-2, and an 11-fold increase in DCs. These data suggest that cellular differentiation is a regulator of local AAT production.

Immunohistochemical analyses of the kinetics and distribution of macrophages, hepatic stellate cells and bile duct epithelia in the developing rat liver

Non-parenchymal cells in the liver consist mainly of Kupffer cells, hepatic stellate (HS) cells and cholangiocytes. To establish base-line data and clarify the nature, this study investigated immunohistochemically the kinetics of these cell populations in developing liver of F344 rats. Samples were collected from fetuses on days 18 and 20, neonates on days 1, 4, 8, 15, and 21, and adults at weeks 5–35. ED1 (CD68)-positive macrophages showed highest number as early as fetal day 18, and then decreased gradually until adulthood. The numbers of macrophages reacting to ED2 (CD163), OX6 (MHC II), and SRA-E5 (scavenger receptor A, CD204) increased after birth (early neonates), and ED2- and SRA-E5-positive cell numbers were maintained until adulthood, but OX6-positive cell number decreased at late stages of neonates and adulthood. ED2- and SRA-E5-positive cells appeared along sinusoids, indicating Kupffer cells, whereas OX6-positive cells were limited in Glisson's sheath. Vimentin-positive HS cells were seen consistently from fetuses to adulthood. Desmin- and glial fibrillary acidic protein (GFAP)-positive HS cells tended to be seen in fetuses and early stages of neonates. HS cells reacting to α-smooth muscle actin (α-SMA) were not detectable. Cholangiocytes, reacting to cytokeratin 19 and AE1/AE3, began to be seen on fetus day 18 with faint reaction, and interlobular bile ducts were completed in Glisson's sheath by neonatal day 8. This study shows that there are heterogeneous macrophage populations and that HS cells can show various cytoskeletal proteins in rat hepatogenesis.

Changes in phenotypically distinct mucosal macrophage populations may be a prerequisite for the development of inflammatory bowel disease

Previous studies have demonstrated the presence of much more marked macrophage heterogeneity in colonic mucosa affected by the idiopathic inflammatory bowel diseases (ulcerative colitis and Crohn's disease) than in normal mucosa. This study examines the morphology, distribution and phenotypic expression of mucosal macrophage-like cells in biopsies from patients with idiopathic inflammatory bowel disease in comparison with disease control samples from patients with colonic infection or ischaemia. Approximately 80% of macrophage-like cells in histologically normal mucosa co-express the antigens recognized by the monoclonal antibodies RFD1 (an interdigitating cell marker) and RFD7 (a marker for mature tissue macrophages). In idiopathic inflammatory bowel disease, the normal colonic macrophage population is partly replaced by cells staining positively with RFD7 alone, and, to a lesser extent, with RFD1+ dendritic cells. Sections from patients with infections and ischaemia exhibited epithelial HLA-DR positivity and infiltration of the lamina propria by a more heterogeneous population of macrophages than that seen in histologically normal mucosa. However, the displacement of the normal colonic macrophage phenotype by RFD7+ tissue macrophages occurred to a significantly greater extent in idiopathic inflammatory bowel disease than in disease control mucosa. A pathognomonic feature of the ulcerative colitis and Crohn's colitis sections was the clustering of RFD9+ epithelioid cells at the bases of disrupted crypts and adjacent to areas of mucosal damage. It is concluded that a degree of macrophage heterogeneity and macrophage infiltration can occur as a non-specific response to colonic mucosal damage. The distinctive feature of idiopathic inflammatory bowel disease mucosa is the almost complete replacement of the normal colonic mucosal macrophage population by tissue macrophages and epithelioid cells, and this phenomenon may be important in promoting the development of a chronic inflammatory state.

Localization and density of myeloid leucocytes in the periodontal ligament of normal rat molars

The phenotypic distribution and density of macrophage-associated antigen-expressing cells in the periodontal ligament (PDL) of normal rat mandibular first molars was evaluated by immunohistochemistry, and an attempt made to identify dendritic cells (DCs) by immunoelectron microscopy. Cells immunopositive to ED1 (a general macrophage marker) were widely distributed throughout the PDL and were most common around blood vessels. A small number of T lymphocytes and OX62 (anti-veiled cells and γδ T cells)-positive DC-like cells were also found. The relative density of cells immunopositive to ED9 (CD14), OX42 (CD11b), OX6 (anti-class II MHC molecules), ED2 (anti-tissue-resident macrophages), 8A2 (CD11c) and WT.1 (CD11a) varied in the mesial, distal and periapical regions of the distal root and the furcal region. This finding suggests that there are several subpopulations of ED1-positive cells which express various combinations of these markers. Immunoelectron microscopy revealed that a small, but distinct, subpopulation of ED1- and OX6-positive cells did have a DC-like ultrastructure, although the majority of these cells were identified as macrophages. The DC-like cells were characterized by poorly developed lysosomal structures and an absence of phagocytic vesicles. It was concluded that the normal rat PDL is equipped with heterogeneous populations of macrophages with regional variations in density. The DC-like cells may function as antigen-presenting cells.

Role of eicosanoids in regulation of macrophage phagocytic functions by platelet-activating factor during endotoxic shock.

We studied the role of eicosanoids in the regulation of macrophage phagocytic functions by products secreted in heterogeneous populations of macrophages and platelet-activating factor during endotoxic shock. Phagocytic activity depended on the metabolism of arachidonic acid in target macrophages and the ratio between its cyclooxygenase and lipoxygenase metabolites produced by heterogeneous populations of macrophages and affecting target cells. The regulatory effect of platelet-activating factor on phagocytosis was related to its interaction with products of the arachidonic acid cascade. Depending on the quantitative ratio of eicosanoids, platelet-activating factor produced various effects on phagocytic functions of heterogeneous macrophage populations.