Expression of urokinase plasminogen activator receptor on monocytes from patients with relapsing-remitting multiple sclerosis: effect of glatiramer acetate (copolymer 1).

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system in which peripheral blood monocytes play an important role. We have previously reported that patients with chronic progressive MS (CPMS) have significantly increased numbers of circulating monocytes which express the urokinase plasminogen activator receptor (uPAR). In the present study, we examined the expression of uPAR on monocytes in patients with relapsing-remitting multiple sclerosis (RRMS) not currently participating in a clinical trial and in patients with RRMS who were enrolled in a double-blind multicenter clinical trial designed to examine the effect of glatiramer acetate (copolymer 1; Copaxone) on relapsing disease. Patients with CPMS have sustained high levels of circulating uPAR-positive (uPAR(+)) monocytes. In comparison, patients with RRMS displayed variable levels of circulating uPAR(+) monocytes. Mean values for uPAR in patients with RRMS were above those seen for controls but were not as high as those observed for patients with secondary progressive MS. Patients with RRMS in the clinical trial also had variable levels of monocyte uPAR. However, patients in the treatment group displayed lower levels following 2 years of treatment. In both placebo-treated and glatiramer acetate-treated patients, the percentage of circulating uPAR(+) monocytes, as well as the density of uPAR expressed per cell (mean linear fluorescence intensity), increased just prior to the onset of a clinically documented exacerbation. Values fell dramatically with the development of clinical symptoms. uPAR levels in all groups correlated with both clinical activity and severity. Results indicate that monocyte activation is impatient in MS and that glatiramer acetate may have a significant effect on monocyte activation in patients with RRMS.

Endothelial cell activation following moderate traumatic brain injury.

Traumatic brain injury (TBI) initiates a cascade of acute and chronic injury responses which include disturbances in the cerebrovasculature that may result in the activation of the microvascular endothelial development of a dysfunction endothelium. The present study examines endothelial cell (EC) activation in a percussion model of moderate TBI. The criteria for endothelial activation used in these studies was surface expression of a number of markers collectively termed endothelial activation antigens. Temporal induction of the major histocompatibility (MHC) class II molecules, E-selectin (CD62E), vascular cell adhesion molecule (VACM-1) (CD106) as well as altered expression of constitutively expressed intercellular adhesion molecule-1 (ICAM-1) (CD54), the glucose transporter protein (glut-1), the transferrin receptor (tfR) (CD71), and MHC class I molecules was examined at various times following impact. Induction of E-selectin and increased expression of ICAM-1 was seen by 2 h post-impact (PI) and was sustained through 24 h PI. Decreased expression of immunologically reactive glut-1 and tfR was observed by 2-4 h PI and remained low up to 24 h PI. No induction of VCAM-1, MHC class II molecules or altered constitutive expression or MHC class I molecules was seen. Changes in EC activation were observed predominantly at the site of impact and were diminished temporarily. These results indicate that mild concussive injury to the brain results in activation of the endothelium.

Pericyte migration from the vascular wall in response to traumatic brain injury.

Any perturbation of the blood brain barrier, whether from changes in cell physiology or from direct injury, may result in microvascular dysfunction and disease. We examined, at the ultrastructural level, microvascular pericyte responses in a well-defined model of traumatic brain injury in the rat. In areas close to the site of impact cortical pericytes underwent a number of changes within the first hour. Approximately 40% of pericytes migrated from their microvascular location. Migration occurred concomitant with a thinning of the abluminal surface of the basal lamina and an accumulation of the receptor for the urokinase plasminogen activator on the leading surface of the migrating cell. Migrated pericytes appeared viable and remained in a perivascular location in the adjacent neuropil. Nonmigrating pericytes in the same section displayed cytoplasmic alterations and nuclear chromatin changes consistent with a rapid degenerative process.

Endothelial activation following prolonged hypobaric hypoxia.

Prolonged exposure to low oxygen may induce adaptive changes which can be either beneficial or deleterious to cell survival. We examined the effect of prolonged moderate hypobaric hypoxia on CNS endothelial cell (EC) function. Exposure to hypoxia resulted in expression of EC activation markers, the cell surface adhesion proteins intracellular adhesion molecule-1 and E-selectin. Induction of the major histocompatibility complex (MHC) class II molecule as well as increased constitutive expression of the transferrin receptor and the glucose transporter-1 protein was also detected within 24 h of exposure to hypobaric hypoxia. Constitutive expression of the MHC class I molecule increased by 48 h. Expression of most EC activation markers increased with time from 0 to 2 weeks. By 3 weeks of exposure to hypobaric hypoxia, ECs returned to their quiescent state with the exception of sustained expression of E-selectin and elevated glut-1. Little to no significant increase in expression of vascular cell adhesion molecule-1 was seen at any time period.

Role of central nervous system microvascular pericytes in activation of antigen-primed splenic T-lymphocytes.

The cellular constituents of the blood-brain barrier (BBB) must make finely tuned, regulatory responses to maintain microvascular homeostasis. The mechanisms by which this task is accomplished are largely unknown. However, it is thought they involve a series of cross-talk mechanisms among endothelial cells (EC), pericytes (PC), and astrocytes. During inflammation, the BBB is exposed to a number of biological response modifiers including cytokines released by infiltrating leukocytes. The response to inflammatory cytokines may alter the normal regulatory function of EC and PC. These changes may account for some of the pathological findings in central nervous system (CNS) inflammatory disease. Previous studies have shown that PC and EC may have immune potential. We have investigated the response of the PC to a variety of inflammatory cytokines. Primary rat PC constitutively express low levels of intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class I molecule, which can be upregulated in response to the cytokine interferon-gamma (IFNgamma). IFNgamma also induced the expression of MHC class II molecule. After induction of MHC class II molecule, CNS PC acquired the capacity to present antigen to primed syngeneic rat T-lymphocytes. Antigen presentation by PC was comparable to that seen with classic antigen-presenting cells. A small number of primary PC constitutively express low levels of vascular cell adhesion molecule-1 (VCAM-1), which was increased on exposure to tumor necrosis factor-alpha (TNFalpha). Results suggest that CNS PC respond to inflammatory cytokines, are involved in T-lymphocyte activation, and express cell surface adhesion molecules (VCAM-1, ICAM-1) that may provide costimulatory activity. It is likely that CNS PC are important in neuroimmune networks at the BBB.

Role of the CNS microvascular pericyte in the blood-brain barrier.

Pericytes are a very important cellular constituent of the blood-brain barrier. They play a regulatory role in brain angiogenesis, endothelial cell tight junction formation, blood-brain barrier differentiation, as well as contribute to the microvascular vasodynamic capacity and structural stability. Central nervous system pericytes express macrophage functions and are actively involved in the neuroimmune network operating at the blood-brain barrier. They exhibit unique functional characteristics critical for the pathogenesis of a number of cerebrovascular, neurodegenerative, and neuroimmune diseases.

CNS microvascular pericytes express macrophage-like function, cell surface integrin alpha M, and macrophage marker ED-2.

CNS pericytes are an integral part of the blood-brain (BBB), but their function is not well understood. We questioned whether primary cultured CNS pericytes have immune potential. Primary cultured pericytes exhibit phagocytic activity when exposed to fluorochrome-conjugated polystyrene beads and antibody-coated zymosan. Maximum phagocytic activity occurred by 3 hr. Pericytes were found to express the macrophage markers ED-2 and the integrin subunit CD11b (alpha M) in culture as well as on isolated microvessels. Pericytes did not express the macrophage marker ED-1. We confirm the heterogeneity of cultured CNS pericytes with regard to expression of alpha-smooth muscle actin. In conclusion, pericytes express macrophage surface antigens and have the ability to perform at least some immune function. CNS pericytes may therefore have a role in neuroimmune networks at the BBB.

Expression of the activation marker urokinase plasminogen-activator receptor in cultured human central nervous system microglia.

The ability of microglia to migrate through central nervous system (CNS) tissue requires proteolytic degradation of components of the extracellular matrix. Urokinase plasminogen activator (uPA), when bound to its cell surface receptor (uPAR), is an active cell surface protease. uPAR expression has been associated with cell activation. Cultured human microglia express surface uPAR. uPAR expression was found to be associated predominately with spindle- or bipolarshaped microglia. The addition of lipopolysaccharide (LPS) to microglial cultures enhanced the proportion of uPAR expression and shifted cell morphology to the elongated spindle or bipolar shape. When microglia were examined immediately ex vivo, uPAR surface expression could not be detected. Similarly, uPAR transcripts detected by reverse transcription-polymerase chain reaction techniques were found in cultured, but not ex vivo, microglia. Microglia isolated from a patient with multiple sclerosis (MS) displayed a large amount of uPAR+ cells. These cells were predominantly spindle or bipolar in nature. These findings suggest that uPAR surface expression is associated with microglial activation. Surface expression of uPAR and associated cell surface protease activity may provide a mechanism for microglial migration and may be important in the pathophysiology of MS.

Recovery phase of acute experimental autoimmune encephalomyelitis in rats corresponds to development of endothelial cell unresponsiveness to interferon gamma activation.

Activation of the vascular endothelium is important in the development of inflammation. Activated endothelial cells (EC) express surface markers not expressed by quiescent EC. These surface markers augment adhesion reactions and leukocyte migration. We examined microvessel EC activation longitudinally in experimental autoimmune encephalomyelitis (EAE) in Lewis rats. CNS microvessels were isolated at 0, 3, 7, 12, 20, and 30 days post-inoculation (PI). Normal and CFA-injected rat microvessels do not express activation antigens (Ag). Increased expression of major histocompatibility complex (MHC) class II molecule and intercellular adhesion molecule-1 (ICAM-1) were detected on CNS microvessels from immunized rats at 7 days PI, prior to development of clinical signs, and at 12 days PI. Enhanced MHC class I molecule was seen only at 12 days. MHC class II molecule expression was focally expressed along microvessel fragments. By 20 days PI, EC did not exhibit increased levels of any of the markers tested. Perivascular cells (possibly pericytes), however, were found to express MHC class II molecule and ICAM-1 up to 30 days PI. During the recovery phase isolated CNS microvessels from MBP-immunized rats were unresponsive to IFN gamma-mediated endothelial activation. Unresponsiveness was independent of IFN gamma concentration. These results suggest that the endothelium is restored to functional quiescence during the recovery phase of acute EAE.

Circulating, soluble adhesion proteins in cerebrospinal fluid and serum of patients with multiple sclerosis: correlation with clinical activity.

Soluble adhesion protein intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule (E-selectin) were measured in serum and cerebrospinal fluid (CSF) of patients with relapsing-remitting multiple sclerosis (RRMS) in remission and in exacerbation, as well as patients with chronic progressive MS, stable MS, and in patients with other neurological and inflammatory diseases (ONDs). Serum ICAM-1 and E-selectin were significantly elevated in patients with MS over those with ONDs and controls. CSF VCAM-1 and E-selectin were found to be elevated over control and disease control samples. No increase in CSF ICAM-1 was observed. Results were analyzed longitudinally and by MS category. In paired CSF and serum samples from patients in exacerbation, elevated VCAM-1 correlated with increased serum VCAM-1 in 5 of 7 patients. Elevated CSF E-selectin did not correlate with elevations in serum E-selectin.

Role of cytoskeletal elements in expression of monocyte urokinase plasminogen activator receptor, activation-associated antigen Mo3.

Peripheral blood monocytes exposed to bacterial products, phorbol esters, cyclic AMP, and cyclic AMP analogs express cell surface activation protein Mo3, which is the human urokinase plasminogen activator receptor (uPA-R). uPA-R is expressed by circulating monocytes from patients with multiple sclerosis (MS). We examined the role of cytoskeletal elements in the surface expression and subcellular distribution of uPA-R in nonactivated and lipopolysaccharide-activated monocytes and in monocytes from patients with MS. By using immunofluorescence techniques and confocal laser microscopy, we found that in unactivated monocytes, cytoplasmic uPA-R is found to one side of the nucleus, colocalizing with the Golgi. Upon activation with lipopolysaccharide, cytoplasmic Mo3-uPA-R becomes dispersed throughout the cytoplasm and projections concomitant with an increase in the monocyte perimeter (spreading). Cytoplasmic dispersion, as well as cell surface deposition, is dependent on microtubule integrity. Cell surface deposition of uPA-R upon activation is reduced by colchicine, which disrupts microtubules; however, once associated at the cell surface, uPA-R becomes associated with microfilaments via vinculin. Disruption of microfilaments with cytochalasin also alters surface expression of immunologically reactive uPA-R, as well as the distribution pattern. Monocytes from patients with MS display the uPA-R distribution pattern characteristic of an activated monocyte.

Cytokine-mediated activation of cultured CNS microvessels: a system for examining antigenic modulation of CNS endothelial cells, and evidence for long-term expression of the adhesion protein E-selectin.

Much of what is known of endothelial responses to cytokines has been derived from in vitro studies using cultured human umbilical vein endothelial cells (EC). Less is known of CNS EC responses and whether intact endothelium responds similarly to cultured cells. We have used techniques by which rat CNS microvessels can be isolated, then cultured in vitro, to study the response of intact endothelium to activation with cytokines. These microvessels are composed of viable EC and perivascular cells, predominantly pericytes. Expression of EC activation antigens in multicellular systems such as cultured microvessels can be assessed quantitatively using immunofluorescence laser cytometry. Interferon gamma increased immunologically reactive major histocompatibility complex class II antigens (< 300 to 2,398 +/- 225 average fluorescence intensity), while tumor necrosis factor alpha induced an increase in vascular cell adhesion molecule-1 (2,167 +/- 171) and E-selectin (1,628 +/- 315). CNS EC appeared to respond similarly to cultured EC with the exception that E-selectin expression was not transiently expressed but was maintained by microvessel EC for 24 and 48 h. Cultured CNS microvessels provide a good system for studying EC activation.

Transforming growth factor beta 1 inhibits cytokine-induced CNS endothelial cell activation.

Postcapillary endothelium at the sites of inflammation undergoes a series of changes collectively termed endothelial cell activation. Activated endothelium expresses immunologically relevant surface proteins that include MHC class II antigens (Ags) and adhesion proteins, as well as exhibits a number of functional changes. Endothelial activation has not been thoroughly studied in CNS endothelium. We have examined cytokine-mediated endothelial activation in isolated rat CNS microvessels. Freshly isolated rat CNS microvessels are viable in culture for at least 72 h. Untreated microvessels express no endothelial activation antigens, but do exhibit constitutive expression of the transferrin receptor (tfR). INF gamma induces a dose-dependent increase in both MHC class II antigens and tfR measured by immunofluorescent staining and quantitated by laser cytometry. IFN gamma-mediated endothelial cell activation could be inhibited with as little as 2 ng/mL TGF-beta 1. although 100% inhibition was seen with 10 ng/mL TGF-beta 1. Cytokine-preactivated endothelial expression of class II Ag and tfR could also be inhibited by TGF-beta 1. TGF-beta 1-treated microvessels become anergic to IFN gamma stimulation. Results suggest that TGF-beta 1 may have a regulatory role in endothelial activation.

Expression of immunologically relevant endothelial cell activation antigens on isolated central nervous system microvessels from patients with multiple sclerosis.

Activation of the vascular endothelium is thought to be an important facet of inflammation, thrombosis, and vasculitis. Activated endothelial cells express a number of immunologically relevant surface markers not expressed by normal endothelial cells. Many of these surface antigens are thought to augment adhesion reactions and migration. Our results show that endothelial activation may play a central role in the pathogenesis of multiple sclerosis (MS). Normal human central nervous system microvessels isolated from autopsy material do not express endothelial cell activation markers, including the adhesion proteins vascular cell adhesion molecule-1 (VCAM-1) and endothelial cell leukocyte adhesion molecule-1 (E-selectin/ELAM-1). They exhibit little to no constitutive expression of immunoreactive intercellular adhesion molecule-1 (ICAM-1) or the urokinase plasminogen activator receptor. Control microvessels exhibit no major histocompatibility complex (MHC) class II antigen. MS microvessels express significant levels of MHC class II antigens, ICAM-1, VCAM-1, and urokinase plasminogen activator receptor. E-selectin was expressed by 3 of 5 MS brains tested. Histologically unaffected areas of MS brain expressed less VCAM-1, ICAM-1, and E-selectin than did microvessels from periplaque zones. However, MHC class II antigens and urokinase plasminogen activator receptor were increased in areas exhibiting little to no evidence of leukocyte infiltration. When microvessels were examined for dual expression of activation markers, we found that in periplaque areas, 50% of microvessels coexpressed HLA-DR and VCAM-1, 28% of microvessels coexpressed HLA-DR and urokinase plasminogen activator receptor, and 43% of microvessels coexpressed HLA-DR and ICAM-1.

Expression of endothelial cell activation antigens in microvessels from patients with multiple sclerosis.

Although the mechanisms governing EC activation are not well understood, evidence points to a role for locally released cytokines from activated leukocytes. We propose that the sequence of events that result in EC activation are important in perivascular leukocyte infiltration into the CNS seen in MS. In the present study we examined expression of EC activation antigens on cerebral microvessels from patients with MS using immunofluorescence staining and quantitation by laser cytometry. Normal human microvessels do not express MHC class II antigens (Ags), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or the urokinase plasminogen activator receptor (uPA-R). They express low levels of transferrin receptors and express factor VIII. Microvessels prepared from MS brain with plaque involvement expressed decreased factor VIII and increased transferrin receptors (tfR). Expression of the adhesion molecules VCAM-1, and ICAM-1 were found on 80% of isolated microvessels. HLA-DR Ags were expressed on 40-60% of microvessels, and the uPA-R was expressed on 50% of MS microvessels examined. MHC class II Ags co-express with VCAM-1 and ICAM-1 more frequently than with the uPA-R. Results indicate that activation of EC in MS is likely to be an important factor in disease pathology.

Expression of monocyte activation antigen Mo3 on the surface of peripheral blood monocytes from patients with multiple sclerosis.

Exposure of human peripheral blood monocytes (PBM) to phorbol esters, bacterial products, and cyclic adenosine monophosphate agonists is known to stimulate expression of a plasma membrane antigen ([Ag]; Mo3). Mo3 is recognized by two monoclonal antibodies, Mo3e (IgM), and Mo3f (IgG). Surface Mo3 is barely detectable by indirect immunofluorescence flow cytometry in nonstimulated monocytes. Mo3-positive monocytes have been found in inflammatory tissues, but increased surface expression of Mo3 in PBM has not been seen in any patient group. We report that PBM from patients with chronic progressive MS (CPMS) express increased Mo3. PBM from patients with other neurologic diseases and healthy controls express little measurable Mo3. No difference was seen in class II major histocompatibility complex Ag expression and in Mo2 (CD14) expression. Exposure of PBM to lipopolysaccharide (10 mg/ml) enhanced Mo3 expression in both MS patients and controls. Mo3 expression on CPMS PBM was not dependent on culture conditions. Taken together, our observations suggest that monocytes from patients with MS are stimulated in vivo to express activation Ag Mo3, but that Mo3-positive monocytes need not be upregulated for HLA-DR.

Production of soluble autocrine inhibitory factors by human glioma cell lines.

Gliomas in vitro exhibit density-limited growth upon the attainment of confluency, an effect usually attributed to cell-cell contact inhibition. Since gliomas have been demonstrated to secrete an array of soluble factors which can enhance tumor growth, we undertook this study to ascertain whether production of soluble factors by the tumor may also inhibit growth in an autocrine manner, and whether production of such factors is associated with the growth phase of the glioma. We observed that cell-conditioned medium (supernatants) from non-confluent glioma cultures induced growth, while confluent culture supernatants produced pronounced growth suppression. These latter supernatants enhanced proliferation of non-transformed astrocytes. Supernatants derived from all stages of confluency produced inhibition of lymphocyte proliferation. To characterize these factors, dialyzed supernatant was tested and found to continue to produce lymphocyte suppression but no glioma growth limitation. Growth of tumors in indomethacin or in acetylsalicylic acid to abolish prostanoid synthesis abrogated the inhibitory influence on glioma growth but only partially reversed the lymphocyte suppressive capacity. These studies suggest that gliomas do produce a growth phase dependent autocrine inhibitory factor(s), and that the production of these small molecular weight factors is at least partially under control of the cyclooxygenase pathway.

Functional desensitization of monocytes from patients with multiple sclerosis due to prostaglandin E.

We previously showed that monocytes from patients with multiple sclerosis (MS) produce increased prostaglandin E (PGE) in tissue culture. In this paper we show that monocytes from patients with chronic progressive MS are less sensitive to agents which stimulate PGE synthesis than healthy controls. MS cells are equally sensitive to PG inhibitors. MS monocytes have increased levels of cellular cyclic AMP and are less sensitive to PGE-mediated increases in cAMP. Depressed monocyte natural cytotoxicity was found to be correlated with cellular cAMP. Functional desensitization of monocytes and decreased sensitivity to PGE signals may be related to chronic exposure to prostaglandin E.

Malignant glioma modulation of immune function: relative contribution of different soluble factors.

We analyzed a series of human glioma cell lines with regard to establishing what variables may contribute to their overall functional immunomodulating capability. We observed that supernatants derived from the gliomas, but not those from non-malignant human astrocyte cultures, suppressed lymphocyte proliferation. The extent of suppression elicited differed between tumors and for the same tumor depending upon its growth phase. For individual gliomas, supernatants from cultures approaching or at confluency elicited maximal lymphocyte suppression. For the series of tumors, levels of production of the immunosuppressive molecules transforming growth factor beta 2 and prostanoids (prostaglandin E2) did not correlate with the levels of functional suppression observed at any of the different growth phases. In some cases, glioma cultures grown in the presence of indomethacin to abolish prostanoid synthesis resulted in supernatants with net stimulatory activity. Our results indicate that malignant transformation of astrocytes is associated with acquisition of immunosuppressive capability which is determined by the combined effect of multiple immunomodulatory soluble factors, inhibitory or enhancing, and is dependent on the growth phase of the tumor.

Cerebrospinal fluid eicosanoid levels: endogenous PGD2 and LTC4 synthesis by antigen-presenting cells that migrate to the central nervous system.

We analyzed CSF from patients with multiple sclerosis, patients with other neurologic diseases, and healthy controls for the presence of prostaglandin (PG) E2, F2 alpha, D2, I, A, and leukotriene (LT) C4. Control CSF had little measurable PGs or LTs. CSF eicosanoids from patients with progressive MS were increased. We found PGD2 only in MS CSF. CSF monocytes from patients in active disease produced significantly increased PGD, PGE, and LTC4 than paired peripheral blood monocytes and monocytes from healthy controls. We saw no significant difference in LTC4 production between MS and control peripheral blood monocytes.