Jun-Ichi Masuyama

Induction of Monocyte Chemoattractant Protein-1 Synthesis in Human Monocytes During Transendothelial Migration In Vitro

Monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) plays important roles in the recruitment of monocytes and thus in the development of atherosclerosis. In this study, we determined whether MCP-1 synthesis was induced by the cellular interaction between monocytes and endothelial cells during the process of transendothelial migration. We found that when human peripheral blood monocytes (2.5 x 10(6) cells) and umbilical vein endothelial cells (HUVECs; 5.0 x 10(5) cells) were cocultured for 5 hours, 7.9 ng/mL MCP-1 was secreted into the medium, whereas when the two were cultured separately, MCP-1 levels were 1.0 and 0.9 ng/mL, respectively. Furthermore, the use of interleukin-1 beta (IL-1 beta)-pretreated HUVECs in cocultures induced twice the levels of MCP-1 as in unstimulated HUVEC culture. Conditioned medium had transendothelial chemotactic activity for monocytes, and this activity was completely abolished by addition of anti-MCP-1 antibody. Although MCP-1 mRNA levels were very low or undetectable in HUVECs or monocytes alone, message could be detected after 2 hours of coculture in total mRNA preparations from both monocytes and HUVECs. mRNA levels increased by 4 hours and had declined slightly by 24 hours. The rapid induction of message suggests that cell contact between monocytes and HUVECs induces the de novo synthesis of MCP-1 protein. Anti-interleukin (IL)-1 alpha/beta and anti-tumor necrosis factor-alpha antibodies, or anti-lymphocyte function-associated antigen-1 and very late antigen-4 antibodies, had little or no inhibitory effects on MCP-1 secretion by cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Monocyte–Endothelial Cell Interaction Induces Synthesis of Granulocyte-Macrophage Colony-Stimulating Factor

BACKGROUND Adhesion of monocytes to the endothelium is an initial step in the early stages of atherosclerosis and inflammation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates a range of functional activities of monocytes, including regulation of monocyte adhesion and induction of cytokine production. We investigated in this study whether CM-CSF synthesis was induced by the direct cell-to-cell interaction between human monocytes and human umbilical vein endothelial cells (ECs). METHODS AND RESULTS The expressions of GM-CSF mRNA and protein were analyzed by Northern blotting and ELISA, respectively. Coculture of monocytes and ECs induced the high levels of GM-CSF mRNA expression, whereas culture of ECs or monocytes alone or coculture of neutrophils with ECs induced no GM-CSF mRNA expression. A large amount of GM-CSF was secreted into the supernatant upon coculture of monocytes with ECs. The supernatant from the coculture markedly stimulated 02- release in neutrophils, and this effect was significantly inhibited by anti-GM-CSF antibody (Ab). Immunohistochemistry and in situ hybridization revealed that GM-CSF protein and mRNA were clearly detectable in both ECs and monocytes adhered to ECs but not in nonadherent monocytes. The GM-CSF production by the coculture was markedly inhibited by genistein and partially inhibited by Abs against interleukin-1 and tumor necrosis factor-alpha. CONCLUSIONS The present results indicate that GM-CSF is produced by direct interaction between monocytes and ECs and suggest that GM-CSF produced locally by monocyte-EC adhesive interaction plays an important role in the pathogenesis of atherosclerosis and inflammation by modulating monocyte/macrophage functions in vivo.

Endothelin-1 release from cultured endothelial cells induced by sera from patients with systemic lupus erythematosus.

OBJECTIVES--To clarify the pathophysiological role of endothelin-1 (ET-1) in the vascular injury associated with systemic lupus erythematosus (SLE) by investigating the effect of sera from patients with SLE on ET-1 release from cultured human umbilical vein endothelial cells. METHODS--Confluent monolayers of cultured human umbilical vein endothelial cells were incubated with serum samples (diluted 1:10) from 25 patients with SLE and 16 normal controls for two hours at 37 degrees C and ET-1 concentration in the culture supernatant was measured by enzyme immunoassay. RESULTS--The mean release of ET-1 from endothelial cells in the presence of serum from SLE patients was greater than in the presence of serum from normal controls (p < 0.005). ET-1 release from endothelial cells significantly correlated with the titre of IgM anti-endothelial cell antibodies (IgM-AECA) and immune complex concentration in sera from SLE patients (p < 0.05 and p < 0.01, respectively). After gel chromatography of the serum from an SLE patient, those fractions containing IgM-AECA or immune complex were shown to stimulate ET-1 release from endothelial cells. Heat aggregated IgG also stimulated ET-1 release from endothelial cells in a concentration dependent manner. CONCLUSIONS--IgM-AECA and immune complexes may stimulate ET-1 release from endothelial cells and ET-1 may play an important role in the initiation and development of vascular injury, such as pulmonary hypertension and lupus nephritis, in SLE.

Monocyte-endothelial cell interaction induces expression of adhesion molecules on human umbilical cord endothelial cells

OBJECTIVE The adhesive interaction of monocytes and endothelial cells has been implicated as a regulatory signal in the cell activation that is involved in the pathogenesis of atherosclerosis. We investigated the effect of monocyte-endothelial cell interaction on the expression of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in human umbilical cord vein endothelial cells (HUVECs). METHODS ICAM-1 and VCAM-1 protein and mRNA expression were determined by cellular ELISA and Northern blot analysis, respectively. RESULTS The addition of unstimulated human monocytes, as well as interleukin-1 beta (IL-1 beta: 25 U/ml) and tumor necrosis factor-alpha (TNF: 100 U/ml), to HUVECs rapidly induced the expression of ICAM-1 and VCAM-1 protein and mRNA in HUVECs, whereas the addition of polymorphonuclear leukocytes (PMNs) had no significant effect on their expression. The induction of ICAM-1 and VCAM-1 by the co-culture of HUVECs and monocytes was significantly, but partially, inhibited by the combination of anti-IL-1 alpha, anti-IL-1 beta and anti-TNF Abs. Actinomycin D and genistein, but not calphostin C, also significantly inhibited the co-culture-induced adhesion molecule expression. CONCLUSIONS These results suggest that the monocyte-endothelial cell interaction induces the expression of ICAM-1 and VCAM-1 in endothelial cells partially through the production of IL-1 and TNF. These findings also suggest that the monocyte-endothelial interaction further augments their interaction through the up-regulation of endothelial adhesion molecules, as a positive feedback mechanism.

Nocardia arthritidis sp. nov., a New Pathogen Isolated from a Patient with Rheumatoid Arthritis in Japan

ABSTRACT Two different bacterial strains with different drug susceptibilities were isolated from the sputum and an inflammatory discharge from a swelling in the left thigh of a patient with rheumatoid arthritis. Both bacterial strains were provisionally assigned to the genus Nocardia on the basis of their morphological and chemotaxonomic characteristics and were further studied in order to establish their taxonomic status. One strain (IFM 10034) was identified as Nocardia farcinica on the basis of its physiological characteristics. The other strain, which was designated Nocardia sp. strain IFM 10035T, revealed a unique pattern of phenotypic properties that distinguished it from other representatives of established Nocardia species. Comparative 16S rRNA gene sequence studies of Nocardia sp. strain IFM 10035T also showed that the bacterium was closely related to the species Nocardia beijingensis. Determination of DNA-DNA relatedness, however, indicated that Nocardia sp. strain IFM 10035T could be delineated from N. beijingensis. The genotypic and phenotypic data combined indicated that the bacterium merits description as a new Nocardia species. The name proposed for the new species is Nocardia arthritidis sp. nov., the type strain being IFM 10035T (NBRC 100137T, JCM 12120T, DSM44731T). The present study suggests that Nocardia infections can be caused by multiple species of the bacterium.

Human monocyte-endothelial cell interaction induces platelet-derived growth factor expression

OBJECTIVE The purpose of this study was to investigate whether the synthesis of platelet-derived growth factor (PDGF), a major mitogen and chemoattractant for vascular smooth muscle cells, was induced by the direct cell-to-cell interaction between human monocytes and umbilical vein endothelial cells (ECs). METHODS PDGF protein and mRNA expression were determined by cellular ELISA, immunohistochemical and Northern blot analyses. RESULTS Coculture of monocytes and ECs secreted a large amount of PDGF into the supernatant, whereas culture of ECs or monocytes alone induced low levels of PDGF production. In Northern blot analysis, substantial amounts of PDGF-A and -B mRNA were induced by coculture of monocytes with ECs. Immunohistochemistry revealed that PDGF-B chain protein was detectable in both ECs and monocytes. PDGF production by ECs induced by conditioned medium of the coculture was significantly inhibited by Abs against interleukin-1 beta (IL-1 beta) and tumor necrosis factor- alpha (TNF alpha). CONCLUSIONS These results indicate that the direct cell-to-cell interaction between human monocytes and ECs induces PDGF synthesis in both types of cells, suggesting that PDGF produced locally by monocyte-EC adhesive interaction plays an important role in the pathogenesis of atherosclerosis by promoting the migration and accumulation of vascular smooth muscle cells.

Subarachnoid hemorrhage and systemic lupus erythematosus

The frequency, clinical profile, treatment and outcome of subarachnoid hemorrhage (SAH) in patients with systemic lupus erythematosus (SLE) were assessed retrospectively, based on the case records of SLE of the Jichi Medical School Hospital over a 20 year period. Clinically defined SAH was found in 10 (3.9%) out of 258 SLE patients, which represented a frequency higher than previously assumed. Five patients had active SLE and lacked an apparent cause of SAH, other than SLE. A high mortality rate (5=5), no visible aneurysm on angiogram (3=4), and an onset during intractable SLE or after discontinued or no steroid therapy because of medical noncompliance (4=5) were characteristic of patients with active SLE, and thus an earlier successful suppression of SLE, if possible, might have prevented their SAH. In contrast, in the 5 patients with inactive SLE, 2 out of 3 saccular aneurysms were succcessfuly clipped and small bleeding of one patient without aneurysms remitted spontaneously without the need for additional steroid therapy. When one death, which occurred outside of medical care, was excluded, the survival ratio of the hospitalized SAH patients with inactive SLE was significantly better than that with active SLE (3=4 versus 0=5, P ‘ 0.0476). In conclusion, the relatively common occurence of SAH in SLE patients, and a significantly different clinical impact of SAH in respect to active and inactive SLE, were suggested from the results.

Acute acalculous cholecystitis in systemic lupus erythematosus: a case report and review of the literature

A 27-year-old woman with systemic lupus erythematosus (SLE) was found to have acute acalculous cholecystitis. At the time of admission, the patient was not under corticosteroid or immunosuppressive therapy. Computed tomography (CT) and ultrasonography revealed findings in the gall bladder consistent with acute acalculous cholecystitis. Her abdominal pain completely disappeared following corticosteroid therapy, with dramatic improvement in the images of CT and ultrasonography. Six similar cases of SLE complicated with acute acalculous cholecystitis have been reported in the literature and they were all treated surgically by cholecystectomy or cholecystostomy. This is the first case report in which acute acalculous cholecystitis accompanying SLE was treated successfully by corticosteroid without surgical intervention.

A novel costimulation pathway via the 4C8 antigen for the induction of CD4+ regulatory T cells.

CD4+CD25+ regulatory T (Treg) cells naturally occur in mice and humans, and similar Treg cells can be induced in vivo and in vitro. However, the molecular mechanisms that mediate the generation of these Treg cell populations remain unknown. We previously described anti-4C8 mAbs that inhibit the postadhesive transendothelial migration of T cells through human endothelial cell monolayers. We demonstrate in this work that Treg cells are induced by costimulation of CD4+ T cells with anti-CD3 plus anti-4C8. The costimulation induced full activation of CD4+ T cells with high levels of IL-2 production and cellular expansion that were comparable to those obtained on costimulation by CD28. However, upon restimulation, 4C8-costimulated cells produced high levels of IL-10 but no IL-2 or IL-4, and maintained high expression levels of CD25 and intracellular CD152, as compared to CD28-costimulated cells. The former cells showed hyporesponsiveness to anti-CD3 stimulation and suppressed the activation of bystander T cells depending on cell contact but not IL-10 or TGF-β. The suppressor cells developed from CD4+CD25−CD45RO+ cells. The results suggest that 4C8 costimulation induces the generation of Treg cells that share phenotypic and functional features with CD4+CD25+ T cells, and that CD25− memory T cells may differentiate into certain Treg cell subsets in the periphery.

Suppressive Role of Endogenous Endothelial Monocyte Chemoattractant Protein–1 on Monocyte Transendothelial Migration In Vitro

Monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) is thought to play an important role in monocyte infiltration into tissue, but little is known about its effect on monocyte-endothelium interaction. We examined the effect of MCP-1 produced by cytokine-activated endothelial cells (ECs) on monocyte-endothelium adhesion and subsequent transendothelial migration by using a double-chamber vessel model. Unstimulated ECs showed no MCP-1 expression, but exposure to interleukin-1 beta (IL-1 beta, 25 U/mL) induced marked MCP-1 mRNA expression and protein synthesis. When placed in the lower compartment, recombinant human (rh) MCP-1 (100 ng/mL) produced a 1.9-fold and a 2.7-fold increase in adhesion and migration, respectively, compared with a corresponding 51% and 59% decrease when placed in the upper compartment. Migration of monocytes was dependent on a gradient of rh-MCP-1 from the apical to basilar side of the EC layer. Furthermore, a forward gradient of MCP-1 induced adherent cells to increase their subsequent migration, whereas a reverse gradient induced the cells to detach and completely inhibited their subsequent migration. Pretreatment with IL-1 beta for 4 and 24 hours produced a 20% and 63% increase in monocyte migration, respectively. In the presence of anti-MCP-1 antibody, the increase was further enhanced by 52% and 152%, respectively. These results suggest that endogenous endothelial MCP-1, when secreted by IL-1-stimulated ECs, suppresses monocyte migration in the presence of MCP-1 on the basilar side of the EC layer.(ABSTRACT TRUNCATED AT 250 WORDS)