Monocyte attachment to laminin in diabetes mellitus

Monocyte-extracellular matrix interactions have been implicated in atherosclerosis pathophysiology. Laminin, the main basement membrane protein contains cell binding domains that can be cryptic, presented only after protein modification. In the present study we evaluated monocyte attachment to laminin-1 in the presence of ATP. Monocytes were derived from either healthy volunteers or patients with diabetes mellitus type II. For the estimation of monocyte attachment to laminin the myeloperoxidase assay was used. Monocytes derived from diabetic patients, showed an increased ability to attach to laminin (p=0.0055). The presence of ATP increased the attachment of control monocytes to laminin (p = 0.0022). On the contrary, the presence of ATP did not affect the attachment of monocytes derived from diabetic patients to laminin. Our results indicate a modified interaction between monocytes and laminin-1 in diabetes mellitus.     

Monocyte attachment and migration through collagen IV in diabetes mellitus

The interactions between monocytes and extracellular matrix proteins have been implicated in atherosclerosis pathophysiology. In the present study we evaluated monocyte attachment and migration through oxidized and non-oxidized collagen IV. Monocyte attachment was tested on microwells coated with either native or oxidized collagen IV. Monocyte migration through collagen IV was examined on transwells. Monocytes derived from patients with diabetes mellitus showed an increased ability to attach and migrate through collagen IV as compared to those derived from healthy volunteers. Moreover, control monocytes attached to oxidized collagen at a higher degree, while they migrated through oxidized collagen at a lower degree, as compared to the native protein. Our results also showed the involvement of the alpha2 integrin subunit in the above phenomena suggesting a modified interaction between monocytes and collagen IV in diabetes mellitus.     

Laminin-1 is phosphorylated by ecto-protein kinases of monocytes

Monocytes encounter basement membranes and interact with laminins while crossing the vascular barrier. It is known that these cells possess ecto-protein kinase activity on their surface. Several proteins of the extracellular matrix can be phosphorylated by ectokinases. Therefore, it has been hypothesized that monocyte ectokinases could phosphorylate laminins and influence their biological properties. In order to test the above hypothesis, we used intact human monocytes and adenosine triphosphate labeled with radioactive phosphate at the third phosphate ([gamma-32P]-ATP) to phosphorylate laminin-1. Autoradiography after sodium dodecyl sulphate polyacrylamyde gel electrophoresis (SDS-PAGE) electrophoresis indicated phosphorylation of laminin-1 on the beta and/or gamma chains. After phosphorylation, phosphoserine could be detected on Western blots by a specific monoclonal antibody. Phosphorylation was not detected when monocytes were pre-treated with trypsin and was inhibited by a specific ecto-protein kinase inhibitor (K252b). Laminin phosphorylation was also inhibited by heparin, a known inhibitor of casein kinase II and by pretreatment of monocytes by a monoclonal anti-casein kinase II antibody. Heparin binding, cell attachment and proliferation, and monocyte migration were enhanced on the phosphorylated laminin-1 as compared to the non-phosphorylated controls. These data indicate that laminin-1 can be phosphorylated by monocyte casein kinase II type ectokinase. This phosphorylation influences important functions of laminin and therefore could provide an additional means for the interaction of monocytes with basement membranes.     

Cariporide Counteracts Atherosclerosis‐related Functions in Monocytes from Obese and Normal Individuals

Objective: This study aimed to show the effect of high glucose concentrations in combination with a pharmaceutical analog of the Na⁺/H+ antiport inhibitor, cariporide, on scavenger receptor CD36 expression, cell adhesion, and cell migration of human monocytes derived from obese and normal individuals. Research Methods and Procedures: Monocytes were isolated from six healthy obese individuals and six healthy age‐ and sex‐matched controls by use of whole blood Percoll sedimentation and plastic surface monocyte binding. The density of CD36 scavenger receptors on the surface of monocytes was assessed by the use of a fluorescent fluorescein isothiocyanate (FITC)‐linked monoclonal antibody. Transmigration of monocytes through laminin‐1–coated filters was performed on 5‐μm pore Transwell culture inserts. Monocyte attachment to laminin was estimated by a solid phase assay. Results: High glucose concentrations caused an increase in monocytes from normal and obese individuals in the expression of CD36 receptors and positively influenced monocyte migration and adhesion to laminin. Cariporide together with glucose counteracted these effects. The effects of migration and adhesion of monocytes to laminin were specific to glucose, because the effect was significantly higher when monocytes were incubated in the presence of 20 mM of glucose than in the presence of 20 mM of fructose. Monocytes from obese subjects showed greater response than in normal to all of the studied effects, with the highest response in laminin attachment. Discussion: The data of this study suggest that cariporide counteracts atherosclerosis‐related functions through Na⁺/H+ antiport inhibition in monocytes from both normal and obese individuals.     

Increased expression levels of monocyte CCR2 and monocyte chemoattractant protein-1 in patients with diabetes mellitus

Increased monocyte recruitment into subendothelial space in atherosclerotic lesions is one of the hallmarks of diabetic angiopathy. The aim of this study was to determine the state of peripheral blood monocytes in diabetes associated with atherosclerosis. Diabetic patients treated with/without an oral hypoglycemic agent and/or insulin for at least 1 year were recruited (n=106). We also included 24 non-diabetic control subjects. We measured serum levels of monocyte chemoattractant protein (MCP)-1, fasting plasma glucose (FPG), HbA1c, total cholesterol, triglyceride, body mass index (BMI), high sensitivity CRP (hs-CRP) and evaluated CCR2, CD36, CD68 expression on the surface of monocytes. Serum MCP-1 levels were significantly (p<0.05) higher in diabetic patients than in normal subjects. In diabetic patients, serum MCP-1 levels correlated significantly with FPG, HbA1c, triglyceride, BMI, and hs-CRP. The expression levels of CCR2, CD36, and CD68 on monocytes were significantly increased in diabetic patients and were more upregulated by MCP-1 stimulation. Our data suggest that elevated serum MCP-1 levels and increased monocyte CCR2, CD36, CD68 expression correlate with poor blood glucose control and potentially contribute to increased recruitment of monocytes to the vessel wall in diabetes mellitus.     

Increased adhesion to fibronectin and Mo-1 expression by diabetic monocytes

As excessive monocyte adhesion to blood vessel wall could produce endothelial cell injury, we undertook comparative studies on the monocyte adhesiveness in insulin-dependent diabetics with vascular complications (n = 26) and healthy, normal subjects (n = 36). In the diabetic group, the extent of monocyte adhesion on fibronectin- and autologous plasma-coated surfaces was significantly increased compared with that in the control group (p less than 0.01 on both types of surfaces). Monocytes adhesion to the plasma-coated surface, but not to the fibronectin-coated surface, could be inhibited by monoclonal anti-Mo-1 antibody in a dose-dependent manner. For diabetic monocyte adhesion, a higher amount of anti-Mo-1 antibody was required to produce a similar extent of inhibition as observed with control monocytes. This indication of increased Mo-1 expression on diabetic monocytes was further confirmed by analyzing the fluorescence intensity of monocytes labeled with the antibody anti-Mo-1. The results of the present study suggest that diabetic monocytes have increased adhesiveness as the result of increased expression of fibronectin and Mo-1 receptors.     

The α1 subunit of laminin-1 promotes the development of neurons by interacting with LBP110 expressed by neural crest-derived cells immunoselected from the fetal mouse gut

A plasmalemmal protein, LBP110, which binds to the α1 chain of laminin-1, is acquired by the neural crest-derived precursors of enteric neurons after they colonize the gut. We tested the hypothesis that laminin-1 interacts with LBP110 to promote enteric neuronal development. The effects of laminin-1 on neuronal development were studied in cultures of cells immunoselected from fetal mouse gut (E14–15) with antibodies to LBP110 or p75^NTR, a marker for enteric crest—derived cells. No matter which antibody was used, the development of cells expressing neuronal markers was increased three- to fourfold by culturing the cells on a laminin-1—containing substrate. To determine whether this effect of laminin-1 is due to the selective adherence of a neurocompetent subset of precursors, immunoselected cells were permitted to preadhere to poly-D-lysine. Addition of soluble laminin-1 24 h later promoted neuronal but not glial development. The laminin-1 induced increment in neuronal development was abolished both by a peptide containing the sequence of the LBP110-binding domain, IKVAV, and by antibodies to laminin α1 that recognize the IKVAV domain. Neither reagent affected the total number of cells. In contrast, the response to laminin-1 was not affected by control peptides, preimmune sera, or antibodies to laminin β1. Laminin-1 transiently induced the expression of nuclear Fos immunoreactivity; this action was blocked specifically by the IKVAV peptide. These data are consistent with the hypothesis that LBP110 interacts with the IKVAV domain of laminin α1 to promote the differentiation of neurons from enteric crest—derived precursors. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 118–138, 1997     

Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice

Atherosclerosis is a major complication of diabetes. Up to 16 weeks of age, the db/db mouse is insulin-resistant and hyperglycemic and is a good model of Type 2 diabetes. After approximately 16 weeks of age, the mice develop pancreatic beta cell failure that can progress to a Type 1 diabetes phenotype. We have previously shown that glucose increases production of endothelial 12/15 lipoxygenase (12/15LO) products in vitro. In young 10-week-old Type 2 diabetic db/db mice, we found significant elevations in levels of urinary 12/15LO products, 12S-hydroxyeicosatetraenoic acid (12S-HETE) and 13S-hydroxyoctadecaenoic acid (13S-HODE) in vivo compared with C57BLKS/J mice. Using isolated primary aortic endothelial cells (ECs) from db/db mice and WEHI78/24 mouse monocyte cells in static adhesion assays, we found increased WEHI monocyte adhesion to db/db ECs (14 +/- 2 monocytes/field for db/db ECs versus 4 +/- 1 monocytes/field for C57BLKS/J ECs, p < 0.002). Thus, ECs from db/db mice appear to be "pre-activated" to bind monocytes. Analysis of db/db ECs revealed a 2-fold elevation in 12/15LO protein compared with C57BLKS/J EC. To determine that 12/15LO products were responsible for the increased monocyte adhesion observed with db/db ECs, we inhibited expression of murine 12/15LO using either an adenovirus expressing a ribozyme to 12/15LO (AdRZ) or with the 12/15LO inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. Treatment of db/db ECs for 48 h with AdRZ or 4 h with 10 microm cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate significantly reduced monocyte adhesion to db/db endothelium (p < 0.009). Thus, inhibition of the murine 12/15LO in db/db mice significantly reduced monocyte/endothelial interactions. We also found that adhesion of monocytes to diabetic db/db ECs was mediated by interactions of alpha4beta1 integrin on monocytes with endothelial vascular cell adhesion molecule 1 and connecting segment 1 fibronectin and interactions of beta2 integrins with endothelial intercellular adhesion molecule 1. In summary, regulation of the 12/15LO pathway is important for mediating early vascular changes in diabetes. Modulation of the 12/15LO pathway in the vessel wall may provide therapeutic benefit for early vascular inflammatory events in diabetes.     

Vitamin D Suppression of Endoplasmic Reticulum Stress Promotes an Antiatherogenic Monocyte/Macrophage Phenotype in Type 2 Diabetic Patients

Cardiovascular disease is the leading cause of morbidity/mortality in patients with type 2 diabetes mellitus (T2DM), but there is a lack of knowledge about the mechanism(s) of increased atherosclerosis in these patients. In patients with T2DM, the prevalence of 25-hydroxy vitamin D (25(OH)D) deficiency is almost twice that for nondiabetics and doubles the relative risk of developing cardiovascular disease compared with diabetic patients with normal 25(OH)D. We tested the hypothesis that monocytes from vitamin D-deficient subjects will have a proatherogenic phenotype compared with vitamin D-sufficient subjects in 43 patients with T2DM. Serum 25(OH)D level inversely correlated with monocyte adhesion to endothelial cells even after adjustment for demographic and comorbidity characteristics. Vitamin D-sufficient patients (≥30 ng/ml 25(OH)D) had lower monocyte endoplasmic reticulum (ER) stress, a predominance of M1 over M2 macrophage membrane receptors, and decreased mRNA expression of monocyte adhesion molecules PSGL-1, β₁-integrin, and β₂-integrin compared with patients with 25(OH)D levels of <30 ng/ml. In vitamin D-deficient macrophages, activation of ER stress increased adhesion and adhesion molecule expression and induced an M2-predominant phenotype. Moreover, adding 1,25(OH)₂D₃ to vitamin D-deficient macrophages shifted their phenotype toward an M1-predominant phenotype with suppressed adhesion. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients activated ER stress, accelerated adhesion, and increased adhesion molecule expression. The absence of ER stress protein CCAAT enhancer-binding protein homologous protein suppressed monocyte adhesion, adhesion molecule expression, and the M2-predominant phenotype induced by vitamin D deficiency. Thus, vitamin D is a natural ER stress reliever that induced an antiatherogenic monocyte/macrophage phenotype.     

Effect of C111T polymorphism in exon 9 of the catalase gene on blood catalase activity in different types of diabetes mellitus

Hydrogen peroxide plays a major role in the pathomechanism of diabetes mellitus and its main regulator is enzyme catalase. The blood catalase and the C111T polymorphism in exon 9 was examined in type 1, type 2 and gestational diabetes mellitus. Compared to the control group (104.7 +/- 18.5 MU/l) significantly decreased (p < 0.001) blood catalase activities were detected in type 2 (71.2 +/- 14.6 MU/l), gestational (68.5 +/- 12.2 MU/l) diabetes mellitus and without change in type 1 (102.5 +/- 26.9 MU/l). The blood catalase decreased (p = 0.043) with age for type 2 diabetics and did not change (p>0.063) for type 1, gestational diabetic patients and controls. Blood catalase showed a weak association with hemoglobin A1c for type 1 diabetic patients (r = 0.181, increasing). The mutant T allele was increased in type 1 and gestational diabetes mellitus, and CT+TT genotypes showed decreased blood catalase activity for type 1 and increased activities for type 2 diabetic patients. The C111T polymorphism may implicate a very weak effect on blood catalase activity in different types of diabetes mellitus.     

The markers of inflammation and endothelial dysfunction in correlation with glycated haemoglobin are present in type 2 diabetes mellitus patients but not in their relatives

The aim of this study is to test several biomarkers of inflammation, of endothelial dysfunction, glycated haemoglobin, and their reflection in arterial dilatation, in patients with type 2 diabetes mellitus and in their relatives, in order to demonstrate if relatives present markers as a form of precocious indicators of diabetes mellitus. Individuals between 30 and 55 years of age and without clinical arterial disease were divided in three groups: type 2 diabetes mellitus patients without complications (12 men and 18 women); first degree relatives of type 2 diabetes mellitus (14 men and 20 women); and control individuals (9 men and 16 women). Body composition was measured with a bioelectrical impedance analyzer and endothelial function with an eco-Doppler device. We determined glucose, insulin, C-peptide, glycated haemoglobin, fibrinogen, E-selectin, P-selectin, soluble intercellular cell adhesion molecule-1 (ICAM-1), soluble vascular cell adhesion molecule-1 (VCAM-1), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP) in plasma. We also studied endothelium independent dilatation and endothelium dependent dilatation. The results: ICAM-1 and VCAM-1 were significantly higher in the diabetic group (237.5+/-43.4 and 692.5+/-168.6 ng/l) than in controls (197.4+/-51.2 and 573.5+/-121.1 ng/l, p=0.011 and 0.013, respectively), but were not higher in the family group (224.5+/-45.2 and 599.8+/-150.4 ng/l). CRP was higher in the diabetic group (3.35+/-3.27 mg/l) than in the other groups (1.28+/-1.29 and 1.61+/-1.54 mg/l, p=0.002) and correlated with glycated haemoglobin. The non-endothelium mediated dilatation was lesser in the diabetic group than in the family group (17.3+/-6.1 vs. 24+/-8, p=0.029) and controls. In conclusion patients with uncomplicated type 2 diabetes, but not their relatives, have biochemical markers of sub-clinical inflammation in relationship with glycated haemoglobin and dysfunction of the endothelial cells markers. In these patients endothelium independent dilatation is more affected than endothelium dependent dilatation.     

Non-Classical Monocytes and Monocyte Chemoattractant Protein-1 (MCP-1) Correlate with Coronary Artery Calcium Progression in Chronically HIV-1 Infected Adults on Stable Antiretroviral Therapy

Background

Persistent inflammation and immune activation has been hypothesized to contribute to increased prevalence of subclinical atherosclerosis and cardiovascular disease (CVD) risk in patients with chronic HIV infection. In this study, we examined the correlation of peripheral monocyte subsets and soluble biomarkers of inflammation to coronary artery calcium (CAC) progression, as measured by cardiac computed tomography scan.

Methods

We conducted a longitudinal analysis utilizing baseline data of 78 participants with HIV infection on stable antiretroviral therapy (ART) in the Hawaii Aging with HIV-Cardiovascular study who had available baseline monocyte subset analysis as well as CAC measurement at baseline and at 2-year follow up. Monocyte phenotypes were assessed from cryopreserved blood by flow cytometry and plasma was assayed for soluble biomarkers using antibody-coated beads in a high sensitivity Milliplex Luminex platform. Change in CAC over 2 years was analyzed as the primary outcome variable.

Results

Of all monocyte subsets and biomarkers tested, higher non-classical monocyte percentage (ρ = 0.259, p = 0.022), interleukin (IL)-6 (ρ = 0.311, p = 0.012), and monocyte chemoattractant protein (MCP)-1 (ρ = 0.524, p = <0.001) were significantly correlated to higher 2-year CAC progression in unadjusted Spearman’s correlation. Non-classical monocyte percentage (ρ = 0.247, p = 0.039), and MCP-1 (ρ = 0.487, p = <0.001), remained significantly correlated to 2-year CAC progression, while IL-6 was not (ρ = 0.209, p = 0.120) after adjustment for age, hypertension, diabetes mellitus, total/HDL cholesterol ratio, smoking history, and BMI.

Conclusion

The percentage of non-classical monocytes and plasma MCP-1 levels were independently associated with CAC progression and may be related to the progression of atherosclerosis and increased CVD risk associated with chronic HIV infection on stable ART.     

Differences in the character of interaction of normal and transformed human keratinocytes with laminin isoforms

Laminins constitute a family of heterotrimeric glycoproteins of basement membranes. Laminins promote cell adhesion, migration, growth, and differentiation. So far, at least 12 different isoforms of laminin have been known. However, no sufficient knowledge is available on the nature of cell response on different laminins. The study was aimed to compare adhesive properties of two laminin isoforms, laminin-1 and laminin-2/4, with respect to normal (freshly isolated keratinocytes) and transformed (A-431) human skin cells. We have used the following assays: cell adhesion to the substrate covered with laminin isoformes, interaction of latex beads (D = 1 micron) coated with the same proteins with cells in suspension, and a comparative study of the cytoskeleton structure of cells spread on the immobilized laminins. It was demonstrated that laminin-2/4 is a more effective potent promotor of adhesion for both normal keratinocytes and transformed A-431 cells, compared with laminin-1. A comparison of many attached protein-covered beads allowed to estimate a relative quantity of cell surface receptors to laminin isoforms in different cell types. The relative number of receptors to laminin-2/4 on the keratinocyte surface is 7 times higher than that to laminin-1 after a 30 min incubation with cells, and is 6 times higher after 1 hour. As for A-431 cells, their attachment to laminin-2/4 beads is 5 times higher than that to laminin-1-beads after a 1 min incubation, but as early as after 5 min this distinction disappeared, owing to bead internalization. The presence of a specific receptor to laminin-2/4 but not to laminin-1 on the keratinocyte surface has been suggested. Keratin differences in cytoskeleton organization in normal and transformed skin cells spread on the substrates covered with laminin-1 and laminin-2/4 were demonstrated.     

Plasma inactive renin in patients with diabetes mellitus: effects of standing and the relation to serum protease inhibitor

In order to investigate the mechanisms of increased plasma inactive renin in diabetics with microvascular complications, changes in active and inactive renin with the progress of diabetes mellitus were studied, and effects of standing on inactive renin release and the relationship between plasma inactive renin and serum trypsin or protease inhibitors wee also studied. Inactive renin increased with the aggravation of diabetes mellitus, but active renin didn't show significant changes with the aggravation of diabetes mellitus. Active renin was significantly increased both in the healthy subjects and in the diabetic patients when they were in an upright position, but no significant change was observed in inactive renin. Serum trypsin in diabetics with retinopathy and nephropathy was lower than that in those with no clinical sign of microangiopathy, but the correlation between plasma inactive renin and serum trypsin was not significant. There was a significant correlation between plasma inactive renin and serum alpha 2-globulin (r = 0.52, p less than 0.01). Although plasma inactive renin was not significantly correlated with serum alpha 1-antitrypsin, there was a significant correlation between plasma inactive renin and serum alpha 2-macroglobulin (r = 0.61, p less than 0.01). These results show that the increased levels of plasma inactive renin observed with the development of diabetic microangiopathy are probably related to the altered plasma protein metabolism observed in patients with diabetes mellitus. However, it is not clear whether this altered protein metabolism is related to the conversion from inactive to active renin.     

Abnormal NF-kappa B function characterizes human type 1 diabetes dendritic cells and monocytes

Dendritic cell (DC) differentiation is abnormal in type 1 diabetes mellitus (T1DM). However, the nature of the relationship between this abnormality and disease pathogenesis is unknown. We studied the LPS response in monocytes and monocyte-derived DCs isolated from T1DM patients and from non-T1DM controls. In T1DM patients, late LPS-mediated nuclear DNA binding by RelA, p50, c-Rel, and RelB was impaired as compared with type 2 DM, rheumatoid arthritis, and healthy subjects, associated with impaired DC CD40 and MHC class I induction but normal cytokine production. In TIDM monocytes, RelA and RelB were constitutively activated, and the src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), a negative regulator of NF-kappaB, was overexpressed. Addition of sodium stibogluconate, a SHP-1 inhibitor, to DCs differentiating from monocyte precursors restored their capacity to respond to LPS in approximately 60% of patients. The monocyte and DC NF-kappaB response to LPS is thus a novel phenotypic and likely pathogenetic marker for human T1DM. SHP-1 is at least one NF-kappaB regulatory mechanism which might be induced as a result of abnormal inflammatory signaling responses in T1DM monocytes.     

Impaired reactive oxygen metabolism of phagocytic leukocytes in NIDDM patients. A role for non-enzymatic glycosylation of collagen

Non-enzymatic glycosylation (NEG) of collagen has been previously shown to significantly influence the reactive oxygen metabolism (ROM) of phagocytic cells in healthy subjects. Considering the role of NEG in the pathophysiology of diabetes, we have further analysed the oxidative metabolism of polymorphonuclear cells (PMNs) and monocytes in 23 patients with non-insulin dependent diabetes mellitus in order to better elucidate a possible pathogenic role of NEG of the extracellular matrix in long-term complications of diabetes. Experiments were performed in triplicate on native-collagen and glycated-collagen coated vials, using a chemiluminescence (CL) assay. Results show that PMNs from diabetic patients display a significant increased basal and zymosan-induced CL activity with respect to controls that are not related to the glycation state of the substrate. Conversely, the CL activity of monocytes induced by zymosan shows a decrease in diabetic patients with respect to healthy volunteers (p < 0.05). Moreover, monocyte CL was reduced by the glycated matrix, both in healthy volunteers and in diabetic subjects (p < 0.05 and p < 0.01, respectively). These data highlight a complex role of phagocytic leukocytes in the pathophysiology of extracellular matrix alterations secondary to NEG that are typically present in clinical conditions such as diabetes or ageing. © 1998 John Wiley & Sons, Ltd.     

Isoform-Specific Attachment of Osteoprogenitors to Laminins: Mapping to the Short Arms of Laminin-1

The recruitment of osteoblast progenitors involves their migration and attachment to the sites of bone formation through interactions with matrix proteins. In a time-limited cell attachment assay, coated laminin-1 inhibits the adhesion of most rat calvaria cells but attaches specifically to osteoprogenitors, as quantified by the number of bone colonies (nodules) formed in the cultures. In order to determine the molecular mechanisms involved in osteoprogenitor attachment to laminin-1, we investigated the effects of laminin-5, a N-truncated laminin variant. In contrast to laminin-1, laminin-5 increased (1.5-fold) rat calvaria cell attachment and did not display any specific affinity for osteoprogenitors. In competition experiments on laminin-5, blocking antibodies directed against either the integrin chain beta1 or the C-terminal portion of laminin-5, as well as thermic denaturation of the protein at 80 degrees C, inhibited rat calvaria cell attachment, suggesting the implication of integrin alpha3beta1 binding to the conformation-dependent C-terminal end of laminin-5. Stepwise thermic denaturation did not suppress the anti-adhesive activity of laminin-1, while osteoprogenitor recruitment was abolished after denaturation above 60 degrees C, suggesting that different domains are involved in these two effects. The anti-beta1 antibody further decreased RC cell attachment to laminin-1, providing evidence for concomitant anti-adhesive and beta1-dependent cell attachment activities. Blocking of beta1 integrin subunit did not, however, reduce osteoprogenitor recruitment. Finally, purified elastase digestion fragment E1+, encompassing the N-terminal short arms of laminin-1, reproduced the effects of the complete molecule in the assay, while C-terminal fragment E8 did not display any cell attachment or osteoprogenitor recruitment properties. In conclusion, the anti-adhesive and osteoprogenitor-selective effects of laminin-1 on rat calvaria cell populations are distinct, beta1-integrin-independent properties mapping to the short arms of the molecule and thus not displayed by the truncated laminin-5.     

Neuroinvasion of fluorescein-positive monocytes in acute simian immunodeficiency virus infection

Monocytes and macrophages play a central role in the pathogenesis of human immunodeficiency virus (HIV)-associated dementia. They represent prominent targets for HIV infection and are thought to facilitate viral neuroinvasion and neuroinflammatory processes. However, many aspects regarding monocyte brain recruitment in HIV infection remain undefined. The nonhuman primate model of AIDS is uniquely suited for examination of the role of monocytes in the pathogenesis of AIDS-associated encephalitis. Nevertheless, an approach to monitor cell migration from peripheral blood into the central nervous system (CNS) in primates had been lacking. Here, upon autologous transfer of fluorescein dye-labeled leukocytes, we demonstrate the trafficking of dye-positive monocytes into the choroid plexus stromata and perivascular spaces in the cerebra of rhesus macaques acutely infected with simian immunodeficiency virus between days 12 and 14 postinfection (p.i.). Dye-positive cells that had migrated expressed the monocyte activation marker CD16 and the macrophage marker CD68. Monocyte neuroinvasion coincided with the presence of the virus in brain tissue and cerebrospinal fluid and with the induction of the proinflammatory mediators CXCL9/MIG and CCL2/MCP-1 in the CNS. Prior to neuroinfiltration, plasma viral load levels peaked on day 11 p.i. Furthermore, the numbers of peripheral blood monocytes rapidly increased between days 4 and 8 p.i., and circulating monocytes exhibited increased functional capacity to produce CCL2/MCP-1. Our findings demonstrate acute monocyte brain infiltration in an animal model of AIDS. Such studies facilitate future examinations of the migratory profile of CNS-homing monocytes, the role of monocytes in virus import into the brain, and the disruption of blood-cerebrospinal fluid and blood-brain barrier functions in primates.