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Background: Genome-wide association studies have discovered a link between genetic variants on human chromosome 15q26.1 and increased coronary artery disease (CAD) susceptibility; however, the underlying pathobiological mechanism is unclear. This genetic locus contains the FES (FES proto-oncogene, tyrosine kinase) gene encoding a cytoplasmic protein-tyrosine kinase involved in the regulation of cell behavior. We investigated the effect of the 15q26.1 variants on FES expression and whether FES plays a role in atherosclerosis., Methods and Results: Analyses of isogenic monocytic cell lines generated by CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing showed that monocytes with an engineered 15q26.1 CAD risk genotype had reduced FES expression. Small-interfering-RNA-mediated knockdown of FES promoted migration of monocytes and vascular smooth muscle cells. A phosphoproteomics analysis showed that FES knockdown altered phosphorylation of a number of proteins known to regulate cell migration. Single-cell RNA-sequencing revealed that in human atherosclerotic plaques, cells that expressed FES were predominately monocytes/macrophages, although several other cell types including smooth muscle cells also expressed FES . There was an association between the 15q26.1 CAD risk genotype and greater numbers of monocytes/macrophage in human atherosclerotic plaques. An animal model study demonstrated that Fes knockout increased atherosclerotic plaque size and within-plaque content of monocytes/macrophages and smooth muscle cells, in apolipoprotein E-deficient mice fed a high fat diet., Conclusions: We provide substantial evidence that the CAD risk variants at the 15q26.1 locus reduce FES expression in monocytes and that FES depletion results in larger atherosclerotic plaques with more monocytes/macrophages and smooth muscle cells. This study is the first demonstration that FES plays a protective role against atherosclerosis and suggests that enhancing FES activity could be a potentially novel therapeutic approach for CAD intervention.

Atherosclerosis, the major cause of vascular disease, is an inflammatory process driven by entry of blood monocytes into the arterial wall. LDL normally enters the wall, and stimulates monocyte adhesion by forming oxidation products such as oxidised phospholipids (oxPLs) and malondialdehyde. Adhesion molecules that bind monocytes to the wall permit traffic of these cells. CD14 is a monocyte surface receptor, a cofactor with TLR4 forming a complex that binds oxidised phospholipids and induces inflammatory changes in the cells, but data have been limited for monocyte adhesion. Here, we show that under static conditions, CD14 and TLR4 are implicated in adhesion of monocytes to solid phase oxidised LDL (oxLDL), and also that oxPL and malondialdehyde (MDA) adducts are involved in adhesion to oxLDL. Similarly, monocytes bound to heat shock protein 60 (HSP60), but this could be through contaminating lipopolysaccharide. Immunohistochemistry on atherosclerotic human arteries demonstrated increased endothelial MDA adducts and HSP60, but endothelial oxPL was not detected. We propose that monocytes could bind to MDA in endothelial cells, inducing atherosclerosis. Monocytes and platelets synergized in binding to oxLDL, forming aggregates; if this occurs at the arterial surface, they could precipitate thrombosis. These interactions could be targeted by cyclodextrins and oxidised phospholipid analogues for therapy.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

More than ten million patients worldwide have been diagnosed with coronavirus disease 19 (COVID-19) to date (WHO situation report, 1st July 2020). There is no vaccine to prevent infection with the causative organism, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), nor a cure. In the struggle to devise potentially useful therapeutics in record time, the repurposing of existing compounds is a key route of action. In this hypothesis paper, we argue that the bisbenzylisoquinoline and calcium channel blocker tetrandrine, originally extracted from the plant Stephania tetrandra and utilized in traditional Chinese medicine, may have potential in the treatment of COVID-19 and should be further investigated. We collate and review evidence for tetrandrine's putative mechanism of action in viral infection, specifically its recently discovered antagonism of the two-pore channel 2 (TPC2). While tetrandrine's particular history of use provides a very limited pharmacological dataset, there is a suggestion from the available evidence that it could be effective at doses used in clinical practice. We suggest that further research to investigate this possibility should be conducted., (© 2020 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but can potentially be cleared by immune system cells or lymphatic drainage. In this study we explore the use of magnetism to hold responsive particles at a delivery site, by incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into layer-by-layer (LbL) microcapsules. Microcapsules with SPIONs were rapidly phagocytosed by cells but did not trigger cellular ROS synthesis within 24 hours of delivery nor affect cell viability. In a non-directional cell migration assay, SPION containing microcapsules significantly inhibited movement of phagocytosing cells when placed in a magnetic field. Similarly, under flow conditions, a magnetic field retained SPION containing microcapsules at a physiologic wall shear stress of 0.751 dyne cm -2 . Even when the SPION content was reduced to 20%, the majority of microcapsules were still retained. Dexamethasone microcrystals were synthesised by solvent evaporation and underwent LbL encapsulation with inclusion of a SPION layer. Despite a lower iron to volume content of these structures compared to microcapsules, they were also retained under shear stress conditions and displayed prolonged release of active drug, beyond 30 hours, measured using a glucocorticoid sensitive reporter cell line generated in this study. Our observations suggest use of SPIONs for magnetic retention of LbL structures is both feasible and biocompatible and has potential application for improved local drug delivery.

Background Genome-wide association studies have shown an association between the single-nucleotide polymorphism rs17514846 on chromosome 15q26.1 and coronary artery disease susceptibility. The underlying biological mechanism is, however, not fully understood. rs17514846 is located in the FES Upstream Region ( FURIN ) gene, which is expressed in vascular endothelial cells (ECs). We investigated whether rs17514846 has an influence on FURIN expression in ECs and whether FURIN affects EC behavior. Methods and Results Quantitative reverse transcription-polymerase chain reaction analysis showed that cultured vascular ECs from individuals carrying the coronary artery disease risk allele of rs17514846 had higher FURIN expression than cells from noncarriers. In support, luciferase reporter analyses in ECs indicated that the risk allele had higher transcriptional activity than the nonrisk allele. Electrophoretic mobility shift assays using EC nuclear protein extracts detected a DNA-protein complex with allele-specific differential binding of a nuclear protein. Knockdown of FURIN in ECs reduced endothelin-1 secretion, nuclear factor-κB activity, vascular cell adhesion molecule-1, and MCP1 (monocyte chemotactic protein-1) expression and monocyte-endothelial adhesion and transmigration. A population-based study showed an association of the rs17514846 risk allele with higher circulating MCP1 levels and greater carotid intima-media thickness. Conclusions The coronary artery disease risk variant at the 15q26.1 locus modulates FURIN expression in vascular ECs. FURIN levels in ECs affect monocyte-endothelial adhesion and migration.

The glycocalyx is a dense layer of carbohydrate chains involved in numerous and fundamental biological processes, such as cellular and tissue homeostasis, inflammation and disease development. Composed of membrane-bound glycoproteins, sulfated proteoglycans and glycosaminoglycan side-chains, this structure is particularly essential for blood vascular barrier functions and leukocyte diapedesis. Interestingly, whilst the glycocalyx of blood vascular endothelium has been extensively studied, little is known about the composition and function of this glycan layer present on tissue-associated lymphatic vessels (LVs). Here, we applied confocal microscopy to characterize the composition of endothelial glycocalyx of initial lymphatic capillaries in murine cremaster muscles during homeostatic and inflamed conditions using an anti-heparan sulfate (HS) antibody and a panel of lectins recognizing different glycan moieties of the glycocalyx. Our data show the presence of HS, α-D-galactosyl moieties, α2,3-linked sialic acids and, to a lesser extent, N-Acetylglucosamine moieties. A similar expression profile was also observed for LVs of mouse and human skins. Interestingly, inflammation of mouse cremaster tissues or ear skin as induced by TNF-stimulation induced a rapid (within 16 h) remodeling of the LV glycocalyx, as observed by reduced expression of HS and galactosyl moieties, whilst levels of α2,3-linked sialic acids remains unchanged. Furthermore, whilst this response was associated with neutrophil recruitment from the blood circulation and their migration into tissue-associated LVs, specific neutrophil depletion did not impact LV glycocalyx remodeling. Mechanistically, treatment with a non-anticoagulant heparanase inhibitor suppressed LV HS degradation without impacting neutrophil migration into LVs. Interestingly however, inhibition of glycocalyx degradation reduced the capacity of initial LVs to drain interstitial fluid during acute inflammation. Collectively, our data suggest that rapid remodeling of endothelial glycocalyx of tissue-associated LVs supports drainage of fluid and macromolecules but has no role in regulating neutrophil trafficking out of inflamed tissues via initial LVs., (Copyright © 2019 Arokiasamy, King, Boulaghrasse, Poston, Nourshargh, Wang and Voisin.)

This review proposes that the development of the atherosclerotic plaque is critically dependent on its inflammatory components forming a self-perpetuating and propagating positive feedback loop. The components involved are: (1) LDL oxidation, (2) activation of the endothelium, (3) recruitment of inflammatory monocytes, (4) macrophage accumulation, which induces LDL oxidation, and (5) macrophage generation of inflammatory mediators, which also activate the endothelium. Through these stages, the positive feedback loop is formed, which generates and promotes expansion of the atherosclerotic process. To illustrate this dynamic of lesion development, the author previously produced a computer simulation, which allowed realistic modelling. This hypothesis on atherogenesis can explain the existence and characteristic focal morphology of the atherosclerotic plaque. Each of the components contributing to the feedback loop is discussed. Many of these components also contain subsidiary positive feedback loops, which could exacerbate the overall process., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

High frequency alternating magnetic fields (AMF) have been widely used as a non-invasive method to induce local hyperthermia for antitumor treatment and to efficiently trigger drug release from various carriers. However, few studies have exploited the potential of targeted drug delivery to healthy cells or tissue and the use of low frequency AMF (LF-AMF) for intracellular triggered release. To achieve this goal, doxycycline was delivered with the layer-by-layer (LbL) assembled magnetic microcapsules, and AMF with low frequency (50 Hz) was applied. The low frequency AMF had little effect on morphology of microcapsules, which upon exposure for 360 min caused no significant damage and had the advantage of minimizing heating effects. Nonetheless, microcapsule permeability increased as a function of exposure time when assessed using FITC-dextran (70 kDa) with the number of permeable microcapsules increased from 13.5% (20 min) to 52.8% (360 min). Increased permeability also enhanced in vitro doxycycline release in genetically engineered myoblast cells where EGFP expression is regulated by the tetracycline system, while targeted EGFP expression was observed by magnetically navigating the microcapsules to a site of interest. Upon LF-AMF exposure of 30 min, no cytotoxicity was observed, but intracellular doxycycline release was promoted and enhanced EGFP expression as demonstrated by EGFP fluorescence intensity measurement. This study reveals the possibility of targeted drug delivery and using LF-AMF as a non-cytotoxic intracellular trigger of drug release from microcapsules without alteration in cell viability., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Genome-wide association studies identified ADAMTS7 as a risk locus for coronary artery disease (CAD). Functional studies suggest that ADAMTS7 may promote cellular processes in atherosclerosis. We sought to examine the association between genetic variation at ADAMTS7 and measures of atherosclerosis using histological, angiographic, and clinical outcomes data., Methods and Results: The lead CAD-associated single-nucleotide polymorphism rs3825807 at the ADAMTS7 locus was genotyped. The G allele (reduced ADAMTS7 function) was associated with a smaller fibrous cap ( P =0.017) and a smaller percentage area of α-actin (smooth muscle cell marker) in the intima ( P =0.017), but was not associated with calcification or plaque thickness, following ex vivo immunohistochemistry analysis of human coronary plaques (n=50; mean age 72.2±11.3). In two independent cohorts (Southampton Atherosclerosis Study [n=1359; mean age 62.5±10.3; 70.1% men] and the Emory Cardiovascular Biobank [EmCAB; n=2684; mean age 63.8±11.3; 68.7% men]), the G allele was associated with 16% to 19% lower odds of obstructive CAD (Southampton Atherosclerosis Study: odds ratio, 0.81; 95% confidence interval, 0.67-0.98; EmCAB: odds ratio, 0.84; 95% confidence interval, 0.75-0.95) with similar effects for multivessel, left anterior descending, and proximal CAD. Furthermore, each copy of the G allele was associated with lower angiographic severity Gensini score (Southampton Atherosclerosis Study, P =0.026; EmCAB, P <0.001), lower Sullivan Extent score (Southampton Atherosclerosis Study, P =0.029; EmCAB, P <0.001), and a 23% lower risk of incident revascularization procedures (EmCAB: hazard ratio, 0.76; 95% confidence interval, 0.59-0.98). There were no associations with all-cause mortality or incident myocardial infarction., Conclusions: Genetic variation at the ADAMTS7 locus is associated with several complementary CAD phenotypes, supporting the emerging role of ADAMTS7 in atherosclerosis and may represent a potential drug target., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD risk.

Background: Elevation of plasma high-density lipoprotein (HDL) cholesterol concentration reduces cardiovascular mortality and morbidity. HDLs have been shown to possess acute anti-inflammatory, antioxidant, and antithrombotic properties. We hypothesize that HDL therapy can acutely alter local and systemic manifestations of plaque instability., Methods: Forty patients with early symptomatic carotid disease were randomized to either receive reconstituted HDL (rHDL) 40 mg/kg (n = 20) or placebo (n = 20). Carotid endarterectomies were performed 24 hr later. Plaques were obtained intraoperatively and used for measurement of thrombomodulatory genes expression. Plasma samples were collected before the infusion, 24 and 48 hr later to measure changes in systemic markers of plaque instability., Results: No significant differences were noted in thrombomodulatory genes expression between the 2 groups. Systemic levels of tissue factor, matrix metalloproteinase 9 (MMP-9), and monocyte chemotactic factor-1 (MCP-1) were significantly reduced in the rHDL group. However, the effects on MMP-9 and MCP-1 were abolished in the immediate postoperative period. Although rHDL did not affect plasma interleukin-6 levels 24 hr following the infusion, it prevented the significant postoperative elevation seen in the placebo group., Conclusions: A single infusion of rHDL can acutely alter plasma biomarkers associated with plaque instability and cardiovascular morbidity., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent genome-wide association studies have revealed an association between variation at the ADAMTS7 locus and susceptibility to coronary artery disease (CAD). Furthermore, in a population-based study cohort, we observed an inverse association between atherosclerosis prevalence and rs3825807, a nonsynonymous SNP (A to G) leading to a Ser-to-Pro substitution in the prodomain of the protease ADAMTS7. In light of these data, we sought a mechanistic explanation for this association. We found that ADAMTS7 accumulated in smooth muscle cells in coronary and carotid atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) of the G/G genotype for rs3825807 had reduced migratory ability, and conditioned media of VSMCs of the G/G genotype contained less of the cleaved form of thrombospondin-5, an ADAMTS7 substrate that had been shown to be produced by VSMCs and inhibit VSMC migration. Furthermore, we found that there was a reduction in the amount of cleaved ADAMTS7 prodomain in media conditioned by VSMCs of the G/G genotype and that the Ser-to-Pro substitution affected ADAMTS7 prodomain cleavage. The results of our study indicate that rs3825807 has an effect on ADAMTS7 maturation, thrombospondin-5 cleavage, and VSMC migration, with the variant associated with protection from atherosclerosis and CAD rendering a reduction in ADAMTS7 function., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16(INK4a), p14(ARF) and p15(INK4b) and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs, VSMC proliferation and VSMC content in atherosclerotic plaques. Immunohistochemical examination showed that VSMCs in atherosclerotic lesions expressed p16(INK4a), p14(ARF) and p15(INK4b). Analyses of primary cultures of VSMCs showed that the 9p21 risk genotype was associated with reduced expression of p16(INK4a), p15(INK4b) and ANRIL (P = 1.2 × 10(-5), 1.4 × 10(-2) and 3.1 × 10(-9)) and with increased VSMC proliferation (P = 1.6 × 10(-2)). Immunohistochemical analyses of atherosclerotic plaques revealed an association of the risk genotype with reduced p15(INK4b) levels in VSMCs (P = 3.7 × 10(-2)) and higher VSMC content (P = 5.6 × 10(-4)) in plaques. The results of this study indicate that the 9p21 variation has an impact on CDKN2A and CDKN2B expression in VSMCs and influences VMSC proliferation, which likely represents an important mechanism for the association between this genetic locus and susceptibility to CAD.

Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemokines can enhance monocyte-endothelial adhesion by activating monocyte integrins. Our study assesses the role of chemokines IL-8, MCP-1 and GRO-alpha, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhesion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, chemokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Immunohistochemistry demonstrated the expression of MCP-1, GRO-alpha and their receptors in the endothelial cells and intima of all atherosclerotic lesions. These results suggest that all these chemokines and their receptors can play a role in the adhesion of monocytes to human atherosclerotic plaques. Furthermore, they suggest that these chemokine interactions provide potential targets for the therapy of atherosclerosis.

OxLDL uptake and cholesterol efflux inhibition in macrophages play a key role in atherosclerotic plaque formation, rupture, and thrombotic ischemia. This study investigates genes implicated in OxLDL uptake (CD36, SRA), cholesterol efflux inhibition (adipophilin, ADFP), and inflammatory recruitments of leukocytes (IL-8) in plaque lesion areas (PLAs) compared to nonplaque lesion areas (NPLAs) in human carotid endarterectomy specimens. Gene and protein expressions were assayed using quantitative PCR and quantitative immunohistochemistry. Pearson tests were used to investigate potential correlation between (a) different gene expressions and (b) gene expression and patient's plasma constituents. CD36, SRA, ADFP, and IL-8 were shown to be significantly more expressed in PLA compared to NPLA. In PLA, a significant correlation was observed between CD36, SRA, ADFP, and IL-8 mRNA levels. Moreover, CD36 expression level was significantly inversely correlated to plasma marker ApoAI. The above investigated genes/proteins may play a key role in the maturation of atherosclerotic lesions.

Tachykinins including substance P (SP) are well known to play a role in influencing oedema formation and leukocyte accumulation during tissue insult and inflammation. Cutaneous inflammatory models to characterize a TNFalpha-dependent mechanism where endogenous SP act via the NK1 receptor to promote leukocyte accumulation in the absence of oedema formation were used. We found that TNFalpha induced dose-dependent leukocyte accumulation at 4 h, which returned towards basal levels at 8 h in NK1+/+ mice. This response was absent in both the NK1+/+ mice treated with an NK1 receptor antagonist and NK1-/- mice. At the highest dose IL-6 induced a significant accumulation in NK1+/+ and NK1-/- mice but IL-12 was ineffective. SP induced skin oedema but none of the cytokines did. Either co-injection of SP with low dose of TNFalpha (0.3 pmol/site) or SP previously injected (30 min) to TNFalpha evoked a significant increase in MPO activity when compared with that induced by the cytokine alone. In contrast, SP injected i.d. 3.5 h after TNFalpha failed to produce additive response. Control, but not capsaicin-pretreated rats (to deplete sensory nerves), exhibited a marked increase in MPO activity in response to TNFalpha. Histological analysis showed that TNFalpha caused tissue infiltrate of leukocytes in NK1+/+ mice, whilst leukocytes accumulated at intravascular sites in NK1-/- mice, but did not appear to emigrate, suggesting a defect in trans-endothelial migration. Interestingly, monocytes in addition to neutrophils accumulated 4 h post TNFalpha injection. In conclusion, the NK1 receptor plays a functional role in mediating leukocyte accumulation independently of the historically important NK1 mediated oedema formation. It seems that TNFalpha directly activates sensory nerve in addition to its chemoattractant activity. The NK1 receptor agonist influences the accumulation of monocytes in addition to that of PMN by 4 h, thus revealing an important influence of the NK1 receptor on TNFalpha mediated events in mouse skin.

Objective and Design: To determine the influence of vitamin C supplementation (500 mg, bd, 14 days) on the circulating concentrations of soluble ICAM-1 (a marker of endothelial activation), neopterin (a marker of monocyte activation), and neutrophil elastase (a marker of neutrophil activation) in smokers and non-smokers in a randomised, double-blind, placebo-controlled trial in a hospital setting., Subjects: Twenty smokers (serum cotinine > or = 20 ng ml(-1)) and 20 age- and gender-matched non-smokers (serum cotinine < or = 13.7 ng ml(-1))., Results: At baseline, there was a significant elevation in the concentration of sICAM-1 in smokers (median 247, IQR 199 to 357 ng ml(-1)) compared to non-smokers (median 207, IQR 189 to 227 ng ml(-1); p = 0.014). Vitamin C supplementation did not influence the circulating concentrations of ICAM-1 or neopterin, or leukocyte elastase activity, in smokers, non-smokers, or in the total population., Conclusions: Markers of monocyte and neutrophil activation were not influenced by smoking status in this study population. However, sICAM-1 concentrations were significantly elevated in tobacco smokers, reflecting tobacco-induced vascular activation that is unaffected by Vitamin C supplementation.

Increased nitric oxide (NO) production after burn injury is well established. However, there is little information relating to the reactions that occur as a consequence of NO generation under such circumstances. We have investigated the synthesis and function of NO in a rat model of local cutaneous thermal injury. We show that NO levels are elevated from 3 hours after injury with a concomitant increase in protein nitration. A selective inducible nitric oxide synthase (iNOS) inhibitor (1400W) significantly attenuated NO synthesis, protein nitration, and neutrophil accumulation in this model, but had no effect on edema formation. The results also indicate that NO synthesis and protein nitration occurred independently of neutrophil accumulation because these parameters were unaffected by depletion of circulating neutrophils. 3-Chlorotyrosine, a marker of neutrophil/myeloperoxidase-mediated protein damage was significantly increased from 1 hour after burn. Our observations provide evidence for the involvement of reactive species in the inflammatory response after burn. The use of selective iNOS inhibitors may represent a novel approach for the management of human burn injuries.

1: We have examined the relationship between neutrophil accumulation, NO(*) production and nitrated protein levels in zymosan-mediated inflammation in rat skin in vivo. 2: Rats were anaesthetized and cutaneous inflammation was induced by zymosan (injected intradermally, i.d.). Experiments were carried out up to 48 h, in recovery procedures as appropriate. Assays for neutrophil accumulation (measurement of myeloperoxidase), nitric oxide (assessment of NO(2)(-)/NO(3)(-)) and nitrated proteins (detected by ELISA and Western blot) were performed in skin extracts. 3: The results demonstrate a close temporal relationship between these parameters. Samples were assayed at 1, 4, 8, 24 and 48 h after i.d. injection of zymosan. The highest levels measured of each parameter (P<0.001 compared with vehicle) were found at 4-8 h, with a reduction towards basal levels by 24 h. 4: Selective depletion of circulating neutrophils with anti-neutrophil antibody abolished neutrophil accumulation and protein nitration. In addition substantially decreased NO levels were found. 5: A selective inducible nitric oxide synthase (iNOS) inhibitor, N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W) also significantly reduced neutrophil levels and NO production and substantially inhibited protein nitration. 6: We conclude that the neutrophil leukocyte plays an essential role in the formation of iNOS-derived NO and nitrated proteins in inflammation, in a time-dependent and reversible manner. The NO-derived iNOS also has a role in stimulating further neutrophil accumulation into skin. This suggests a close mechanistic coupling between neutrophils, NO production and protein nitration.

A simple, sensitive, colorimetric labelling method was devised to quantify cell adhesion, based on labelling the cell plasma membrane with biotin. This method was applied in adhesion assays, which involved the adherence of biotin-labelled, PMA-stimulated, U937 cells. These cells resemble monocytes, and were bound onto fibronectin-coated wells and to an ECV304 cell monolayer. The adherent U937 cells were detected by the addition of a peroxidase-conjugated anti-biotin antibody and a soluble colorimetric substrate. This assay is convenient, fast and sensitive, and able to detect 320-1000 U937 cells under the conditions described. This study has used titration assays to compare the biotinylation method with the existing cell quantification approaches of 51Cr radiolabelling and antibody dependent ELISA. Chromium labelling was the most sensitive technique, but we found the biotinylation method to be more convenient than radioactive labelling and more sensitive than conventional ELISA. Biotinylated cells were also used very effectively in a Stamper-Woodruff adhesion assay with U937 cells binding to histological sections of atherosclerotic plaques. The selective detection of the bound cells permitted automated quantitation by image analysis. Whole cell biotinylation may have wider applications in biological research.

Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE(-/-) mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE(-/-)) or double-knockout (DKO; ApoE(-/-), ICAM-1(-/-)) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

There is some evidence to suggest that smoking may affect circulating levels of CD44 (sCD44) molecules. Therefore, we investigated the effect of smoking on the circulating level of sCD44 by comparing the change in total sCD44, sCD44v5, and sCD44v6 concentrations over 1 year in a group of people who quit smoking (n = 30) and a control group of people who continued to smoke (n = 30). Smoking status and compliance were monitored by analysis of plasma cotinine and expired CO levels and also by self-reported tobacco use. We show a dose-dependent relationship between smoke intake and baseline plasma concentrations of reputed tumor-associated CD44 variant isoforms (sCD44v5 and sCD44v6) in smokers (n = 60). There was a significant decline in the level of both sCD44v5 and sCD44v6 in quitters as compared with continuing smokers [-13.2 (95% confidence interval, -7.6 to -18.8; P < 0.001) and -62.2 ng/ml (95% confidence interval, -33.9 to -90.6; P < 0.001), respectively], but not in the total sCD44 concentration. These results show that the increased concentrations of sCD44v5 and sCD44v6 in smokers are dose related and reversible and suggest that the attributed diagnostic specificity and prognostic value of sCD44 molecules in malignant and inflammatory disease may be affected by smoking status.

Soluble adhesion molecules have been reported as risk markers of a wide range of human diseases and specific adhesion molecules may play a direct role in pathological processes. Serum soluble intercellular adhesion molecule-1 (sICAM-1) is known to be significantly elevated in smokers compared to non-smokers. We examined the acute effects of smoking a standard 2R1 research cigarette on the serum concentration of sICAM-1 and other circulating adhesion molecules (sP-selectin, sE-selectin, sL-selectin, sVCAM-1 and sPECAM-1) in heavy smokers (serum cotinine >/= 100 ng/ml), light smokers (serum cotinine

Soluble CD44 isoforms have been reported as markers of specific malignancies and inflammatory diseases. However, recent reports suggest tobacco smoking may lead to an elevation in the circulating concentration of specific CD44 variants. We, therefore, investigated the effect of smoking status on circulating levels of total sCD44. Total soluble CD44 was measured by enzyme-linked immunosorbent assay in the serum of two age- and gender-matched groups consisting of smokers (n = 19) and non-smokers (n = 20). Smoking status was confirmed by analysis of serum cotinine. The concentration of total sCD44 was found to be significantly elevated in smokers compared with non-smokers (p = 0.025). The observation that total sCD44 concentration is raised in smokers may have relevance to the aetiology of smoking-associated diseases. The effect of smoking on sCD44 concentrations should be considered when assessing the role of sCD44 as a marker of inflammatory disease, cancer, or other disease processes.

Tobacco smoking is probably the most important, controllable environmental risk factor in periodontitis. It results in changes in the vascular, inflammatory, immune and healing responses. The degree of exposure to tobacco smoking can be measured in pack years or by measuring serum cotinine and nicotine levels. In a previous paper we reported elevated levels of serum soluble intercellular adhesion molecule-1 (sICAM-1) in smokers, regardless of periodontal status. Elevated sICAM-1 has been found to be a risk marker for cardiovascular disease. In the present paper we report the short-term effects of an episode of smoking on blood flow and levels of sICAM-1. Human volunteers included non-smokers, light smokers and heavy smokers. Relative blood flow was monitored in the gingivae and forehead skin using a laser Doppler flowmeter and serum levels of sICAM-1, cotinine and nicotine measured before during and up to 60 min following an episode of smoking. We could not provide evidence to support the theory that there is localized vasoconstriction within the gingival tissues. In contrast, there was a significant increase in blood flow in the forehead skin of light smokers which was not observed in non-smoking controls or in heavy smokers, suggesting a long-term tolerance in this latter group. The level of sICAM-1 remained unchanged during this episode, further suggesting a long-term effect. In a parallel group of subjects, we were able to demonstrate a direct significant correlation between sICAM and serum cotinine levels. These observations may be relevant to aetiological mechanisms in periodontitis and other smoking-associated diseases.

Background: Accumulating evidence suggests that the cytokine network is central to the immunopathology of bronchial asthma and the existence of naturally occurring cytokine antagonists has added to this complexity. Upregulation of both interleukin 1 beta (IL-1 beta) and its naturally occurring receptor antagonist, interleukin 1 receptor antagonist (IL-1ra), has previously been observed on asthmatic bronchial epithelium compared with normal airways., Methods: The effect of inhaled beclomethasone dipropionate (BDP) on asthmatic bronchial epithelial expression of IL-1 beta and IL-1ra was studied. Frozen bronchial biopsy specimens from nine asthmatic subjects receiving 1000 micrograms BDP daily for eight weeks and from six asthmatic subjects receiving matching placebo were stained with anti-IL-1 beta and anti-IL-1ra antibodies. Hue-saturation-intensity (HSI) colour image analysis was used to quantify the brown immunoperoxidase reaction colour present on the bronchial epithelium., Results: There was a significant twofold decrease in the epithelial expression of IL-1 beta after treatment with BDP but no significant change was seen in IL-1ra (P = 0.175)., Conclusion: The selective inhibition of IL-1 beta, without effect on IL-1ra, provides a novel mechanism for the anti-inflammatory action of glucocorticosteroids.

Accumulating evidence suggests that the cytokine network is central to the immunopathology of bronchial asthma and recent findings have suggested that naturally occurring cytokine antagonists may also be involved. In this study we looked at the expression of interleukin-1 beta (IL-1beta) and its naturally occurring receptor antagonist, IL-1ra, in the normal and asthmatic bronchial wall. Frozen bronchial biopsies from 12 normal and 18 asthmatic individuals were double stained with EBM11 (a CD68 macrophage marker) and either a rabbit anti-IL-1beta or a rabbit anti-IL-1ra. Hue-saturation-intensity color image analysis (HSI) was used to quantify the brown immunoperoxidase reaction product present on the bronchial epithelium. There was an increased expression of both IL-1beta and IL-1ra in the asthmatic bronchial epithelium, p < 0.0002 and p < 0.0001, respectively. Additionally, the numbers of macrophages, of IL-1beta producing cells, and the percentage of macrophages producing IL-1beta were significantly increased in the asthmatic submucosa (p < 0.004, p < 0.002, and p < 0.008, respectively).

Blood-derived macrophages in the arterial intima are a characteristic feature of active atherosclerotic plaques. Adherent monocytes on the luminal surface and increased adhesion molecules on the endothelium have suggested that specific molecular mechanisms are involved in monocyte/macrophage traffic into the arterial wall. Adhesion of human monocytes and related cell lines was therefore studied in vitro to histological sections of human plaques. At 37 degrees C, these cells bound selectively to the plaques. Binding to the endothelium occurred and was also present extensively in the diseased intima. Inhibition studies showed that the endothelial and general intimal binding had largely similar molecular properties. Strong inhibition was produced by antibodies to the monocyte-specific adhesion molecule CD14, to beta2 integrins, and to ICAM-1. Likewise, a peptide containing the Arg-Gly-Asp sequence was strongly inhibitory, suggesting that binding of leukocyte integrins to arterial extracellular matrix was synergistic with cell-cell interactions. A P-selectin antibody was exceptional in giving selective inhibition of endothelial adhesion, which correlates with the specific endothelial localization of this adhesion molecule. These results show that monocytes adhere to atherosclerotic plaques through the focal activation of multiple arterial wall adhesion molecules, confirming the adhesion hypothesis. A positive feedback theory for the pathogenesis of atherosclerosis can be suggested, based on the ability of macrophages in the wall to activate the endothelium, induce adhesion molecules, and facilitate additional monocyte entry. The adhesion assay provides a means for the identification of adhesion inhibitors with therapeutic potential.

We have used the classical tuberculin cutaneous delayed hypersensitivity immune response to investigate the effects of corticosteroids on mononuclear cell function in nine subjects with corticosteroid-resistant asthma (CRA) and six subjects with corticosteroid-sensitive asthma (CSA), who demonstrated sensitivity to intradermal purified protein derivative (PPD) of Mycobacterium tuberculosis. In a double-blind, crossover, placebo-controlled study, patients were given oral prednisolone or placebo, starting on day 0, and a predetermined intradermal dose of PPD on day 7. On day 9 the site of the induration was measured and biopsied for immunohistochemical analysis. There was no difference in skin induration between the CSA and CRA groups during the placebo arm of the study (p = 0.38). Prednisolone significantly suppressed the cutaneous induration (p, 0.003) in the CSA group but not in the CRA group. As compared with placebo, the number of macrophages (p = 0.018), eosinophils (p = 0.009), and T memory cells (p = 0.009) was suppressed by prednisolone in the CSA group but not in the CRA group. No significant suppression of the number of neutrophils or monocytes/immature macrophages was induced by prednisolone in either group. There was no difference in intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 expression in blood vessels or epidermis between the CSA and CRA groups, with no suppression by prednisolone in either group. These findings suggest a defect in the responsiveness of cellular immune mechanisms to the suppressive effects of corticosteroids in steroid-resistant asthma. The differential suppressive effects of corticosteroids on cellular recruitment in the PPD response between the individuals with CSA and those with CRA are not due to modulation of expression of endothelial adhesion molecules.

Objectives: Cytokines, released from mononuclear cells (MNC) are mediators of joint destruction in rheumatoid arthritis (RA). The mechanisms of action of gold salts used in the treatment of RA are unknown. The aim of this study was to investigate cytokine expression and intensity of MNC infiltrate in the RA synovial membrane (SM) following treatment with sodium aurothiomalate (SAT)., Methods: Sequential blind needle biopsies were obtained at entry into the study and at two and 12 weeks after the start of SAT therapy in 10 patients with active RA. SMs were stained with a panel of monoclonal antibodies to assess cytokine expression (IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and GM-CSF)., Results: There was a significant decrease in IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha expression 12 weeks after treatment (p < 0.004, p < 0.002, p < 0.009 and p < 0.004 respectively). This was noted in the lining layer, the perivascular aggregates and the connective tissue areas. Detailed examination of the MNC infiltrate showed a significant reduction in inflammatory monocytes (MONO) in the lining layer at two weeks (p < 0.03). A decrease in the number of CD68+ macrophages (MAC) was noted in the perivascular and connective tissue areas at 12 weeks. No significant changes were observed in the number of T and B cells and blood vessels., Conclusion: The results suggest that gold may suppress RA disease activity by diminishing MONO and MAC numbers and consequently monokine production in the SM.

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis.

The expression of the monocyte chemoattractant protein (MCP-1), a member of the chemokine family of low molecular weight cytokines, was assessed by immunohistochemistry in bronchial biopsies from 12 asthmatic and 12 normal subjects. Both a monoclonal antibody (F9) and a polyclonal antibody were employed to detect MCP-1, while the mouse myeloma protein (MOPC21) was used as a negative control. Strong positive reactions for MCP-1 were seen in the bronchial epithelium. Subepithelial macrophages, blood vessels, and bronchial smooth muscle were also stained. Hue-saturation-intensity color image analysis was used to quantify reactions of the monoclonal antibody in the epithelial and subepithelial layers. With the monoclonal antibody, asthmatic biopsies showed 51.8 +/- 3.7% (mean +/- SEM) of the epithelium staining positively, whereas normal subjects reacted much less, with 6.4 +/- 1.9% of the epithelium staining (P < 0.0001); there was no overlap between the two groups. Likewise, staining was increased in the subepithelium of asthmatic airway biopsies, with 11.5 +/- 3.1% and 2.0 +/- 1.0% staining positively in asthmatic and normal subepithelium, respectively, (P < 0.002). There was a significant correlation between staining of the epithelium and subepithelium (r = 0.77, P < 0.001). The polyclonal anti-MCP-1 antibody also gave strong reactions in the epithelium and subepithelium, with 34.0 +/- 7.8% of the asthmatic and 1.6 +/- 1.0% of the normal bronchial epithelium staining positively (P < 0.0001). These increased levels of MCP-1 in the asthmatic airways suggest that they may play a role in macrophage recruitment and activation and thereby contribute to the inflammatory pathology of bronchial asthma.

We studied the antigenic markers of macrophages (Mphs) in atherosclerotic human arteries by immunohistochemistry and compared them with the patterns in Mph subpopulations of tonsil and lymph node, which are also described. The staining of atheroma intimal Mphs was assessed semiquantitatively in the subendothelial, mid, and outer intima. Three patterns of reactivity with Mph antibodies were recognized. (a) Pan-Mph (antibodies HAM56, EBM11, and CD14 group). Staining was maximal in the mid-intimal zone. (b) Subendothelial Mphs (anti-muramidase, anti-alpha-1-antitrypsin and MAC387). In lymphoid tissue, sinusoidal Mphs and a few inflammatory Mphs were stained, as well as blood monocytes. This group of antibodies recognizes Mphs that are likely to be recently blood-derived (RBD-Mphs). (c) Antibodies reactive with various histiocyte populations in lymphoid tissues (anti-Factor XIII; anti-HLA Class II and LN2) also gave maximal staining in the mid-intimal zone, but differences between lesion types suggest that they are recognizing heterogeneous subpopulations of Mphs. These observations demonstrate the heterogeneity of tissue Mphs and suggest that an insight into the dynamics of tissue Mphs can be obtained from the cell phenotype. They indicate that all stages of atherosclerosis can have an outward traffic of Mphs from the blood through the arterial intima.

The presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in airway epithelial cells in vivo was assessed in 15 asthmatic and 9 normal subjects. GM-CSF was analyzed using immunohistochemistry with a polyclonal and a monoclonal antibody. Hue saturation intensity color image analysis was used to quantify staining. Asthmatic airway epithelial cells stained significantly more with anti-GM-CSF than those from normal subjects (p = 0.0013 and p = 0.0003 for the polyclonal and monoclonal antibodies, respectively). Additionally, 8 asthmatic individuals inhaled 1,000 micrograms beclomethasone diproprionate per day for 8 wk and 6 asthmatic patients inhaled matching placebo. There was a significant reduction of GM-CSF in the epithelium in the patients who were given corticosteroids (p = 0.014), whereas the group of subjects who were given placebo showed no significant change in GM-CSF staining. There was a correlation between the percentage suppression of GM-CSF staining by inhaled corticosteroids and the percentage increase in FEV1 (r = 0.61, p < 0.05) and percentage decrease in carbachol responsiveness (r = 0.80, p < 0.01). These findings suggest that GM-CSF may play a role in the inflammatory processes of bronchial asthma and that the epithelial cell may be a target cell for drug action.

Background: The intercellular adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) has been implicated in a number of interactions between leukocytes and cells of lymphoid and connective tissue, including endothelial cells. Such interactions within synovial tissue may be important in the pathogenesis of rheumatoid arthritis., Experimental Design: The expression of VCAM-1 on specific cell populations in normal and inflamed synovium was investigated using a range of double-labeling techniques., Results: The strongest VCAM-1 staining was found to be confined to four nonmacrophage populations (as judged in terms of CD68 expression, nonspecific esterase activity, content of prolyl hydroxylase and activity of uridine diphosphoglucose dehydrogenase): (i) type B synoviocytes, (ii) vascular wall cells outside the endothelial layer, (iii) scattered stromal cells with cytoplasmic processes, and (iv) cells resembling follicular dendritic reticulum cells in lymphoid aggregates with germinal centers (in the three samples of rheumatoid arthritic tissue where these were present). Some macrophages of the synovial intima showed weak VCAM-1 staining. Endothelial cell staining was seen but it was consistently weaker than the staining of cells of group ii., Conclusions: The four cell populations described as showing bright staining for VCAM-1 may all be involved in local interactions with leukocytes of the macrophage or lymphoid series subsequent to initial leukocyte entry into the tissue. Expression of VCAM-1 by these cells may play a role in such interactions.

Bronchial biopsies obtained from 16 asthmatic patients and six normal subjects were analyzed by immunohistochemistry. In the asthmatic patients, the total numbers of macrophages infiltrating the airway mucosa were increased. Many of the macrophages had the phenotypic characteristics of blood monocytes. HLA Class II antigen was expressed on infiltrating cells and airway epithelial cells. In biopsies from the asthmatics there was a significant increase in activated eosinophils, but not in neutrophils. There was also a significant increase in the numbers of T-lymphocytes in the asthmatics, but very few B-lymphocytes were detected. These results suggest that lung macrophages may have a central role to play in the mechanisms of the chronic immune-mediated inflammatory response seen in the airway mucosa of asthmatic patients.

Immunohistochemistry of human atherosclerotic arteries demonstrates expression of the intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, macrophages, and smooth muscle cells of the plaques. Normal arterial endothelial cells and intimal smooth muscle outside plaques give weaker or negative reactions; these differ from the strong endothelial expression in small vessels. Quantitative color-image analysis of the endothelial layer shows increased expression of ICAM-1 in all subtypes of atherosclerotic lesions, except fibrous plaques. Endothelial expression of ICAM-1 may be involved in the recruitment of monocytes to the lesion, as suggested by its role in the entry of leukocytes, including monocytes, into foci of inflammation. Collaboration with other mechanisms, particularly chemoattractant factors, may be important for this effect. ICAM-1 enhanced monocyte recruitment is a potential mechanism for the growth of an atherosclerotic plaque.

There is increasing evidence implicating the central role of cells of monocyte/macrophage lineage in the pathogenesis of bronchial asthma. This evidence comes from studies on peripheral blood monocytes. BAL fluid and cells and, more recently, airway immunohistochemistry. Elucidation of the mechanisms of macrophage interactions may eventually lead to novel approaches in anti-asthma therapy., (© 1992 S. Karger AG, Basel.)

Endothelial cell adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) are cytokine-regulated cell surface molecules involved in leukocyte adhesion. We have studied two forms of cutaneous inflammation to investigate in vivo the kinetics of adhesion molecule expression in relation to tissue accumulation of leukocytes. Immunohistology was performed on skin biopsies taken from human volunteers at 1, 6, 24, 72 h, and 1 week after two minimal erythema doses (MED) of ultraviolet B (UV-B) or intra-cutaneous tuberculin-purified protein derivative (PPD) (10-100 U). ELAM-1 expression on vascular endothelium and polymorphonuclear leukocyte infiltration were first observed at 6 h and maximal at 24 h after both UV-B and PPD. At 72 h and 1 week, however, endothelial ELAM-1 was more strongly expressed in PPD biopsies. VCAM-1 was minimally expressed in control skin, and was induced above background levels on endothelium, on some perivascular cells, and on stellate-shaped cells in the upper dermis at 24 h after injection of PPD; it was maintained up to 1 week. In contrast, no induction of VCAM-1 was seen following challenge with either 2 or 8 MED UV-B. Following PPD, but not UV-B, there was marked induction of ICAM-1 expression on basal keratinocytes. In these biopsies, the inflammation induced in response to PPD therefore differed from UV-B-induced inflammation in showing prolonged expression of endothelial ELAM-1, induction of VCAM-1 on endothelium and other cells, and induction of keratinocyte ICAM-1. These differences may result from differences in the cytokines released and may in turn be responsible for the differences in the nature of the leukocytic infiltration during the two types of inflammatory response.

We have compared the adhesion of 51Cr-labeled eosinophils and neutrophils to cultured human umbilical vein endothelial cell (EC) monolayers that have been stimulated with IL-1, TNF, or LPS. Each agent stimulated the adhesion to EC of both eosinophils and neutrophils in a similar dose- and time-dependent manner. F(ab')2 fragments of mAb 1.2B6 (anti-endothelial leukocyte adhesion molecule (ELAM)-1) and mAb 6.5B5 (anti-intercellular adhesion molecule (ICAM)-1) each inhibited partially, and to a similar extent, eosinophil and neutrophil adhesion to EC monolayers prestimulated with TNF (10 ng/ml) for 6 h. Greater inhibition of both eosinophil and neutrophil adhesion was achieved by combining the effects of mAb 1.2B6 with either mAb 6.5B5 or mAb TS1/18 (anti-CD18). These observations indicate that both ELAM-1 and ICAM-1 are involved in the adhesion of eosinophils and neutrophils to EC stimulated with TNF. In order to determine whether these molecules are expressed in vivo during allergen-induced late phase allergic responses in the skin, human skin biopsies were examined at 6 h after Ag or saline challenge with the use of an alkaline phosphatase-staining technique. Both ELAM-1 and ICAM-1 were expressed with greater intensities in Ag-challenged biopsies, suggesting that these molecules may be involved in granulocyte recruitment in vivo. The similarities we have established between mechanisms of eosinophil and neutrophil adhesion to cytokine-stimulated EC suggests that factors other than differential leukocyte-EC adhesion may be responsible for the selective accumulation of eosinophils at sites of allergic inflammation.

Using the monoclonal antibody HAM-56 with the avidin-biotin method on recent or archival paraffin-embedded tissue from multiple sclerosis brains, we have been able to distinguish between acute, chronic active and inactive lesions. HAM-56 stains all macrophages, monocytes and at least some microglia; it is the only pan-macrophage marker to our knowledge that can be satisfactorily used on conventional paraffin sections. A much narrower range of mainly perivascular macrophages in acute plaques of multiple sclerosis is stained with MAC-387, anti-muramidase and anti-alpha1-anti-trypsin. The acute plaques show HAM-56-stained macrophages throughout the lesion, and these macrophages exhibit profiles of phospholipid-rich myelinic bodies, which are also usually stainable with Luxol fast blue. Active ongoing lesions show a rim of macrophages at the edge of the lesion. These macrophages show profiles of large vacuoles, thought to represent the sudanophilic esterified cholesterol formed during demyelination. Inactive cases show none of these features; the few perivascular macrophages present often contain the end product of lipid peroxidation, ceroidlipofuscin.

The periventricular region was studied in the brains of 129 cases of multiple sclerosis, with the purpose of establishing the mechanism and order of events in the development of the periventricular plaque, and deciding whether there is any relationship between granular ependymitis and such plaques. Periventricular plaques were found in 82.2% of cases. Observation and computerized morphology showed that the early stage of the periventricular plaque is the formation of a lesion around a subependymal vein and that adjacent lesions later coalesce. These plaques do not appear to arise from the ependyma, which is against any role for the CSF in their initial development. Chronic or burnt-out periventricular lesions often show overlying granular ependymitis (10.9% of cases) and subependymal gliosis (17.8%), presumably as a result of the long-continued low-grade inflammatory process. This process, which is not specific for multiple sclerosis, is sometimes associated with transfer of IgG and C3, as shown with peroxidase methods, across the subependymal vein wall and the ependymal epithelium. Increased permeability of the inflamed ependyma constitutes a possible abnormal entry route from plaque to CSF or, in reverse, from CSF to brain.