TGF-beta completely blocks the formation of small-cell colonies: effects of mito-inhibitory factors on the proliferation of primary cultured rat hepatocytes.

The proliferation of primary cultured rat hepatocytes was observed in serum-free modified DMEM supplemented with 10 mM nicotinamide and 10 ng/ml EGF. These proliferating cells were mainly mononucleate and formed small-cell colonies after 4 days of culture. In the present experiment primary cultured hepatocytes were treated with Activin A, IL-1 beta, IL-6, and TGF-beta, which have been shown to be inhibitors of the DNA synthesis of rat hepatocytes, to examine whether these four inhibitors could suppress the formation of small-cell colonies. The initial DNA synthesis of more than 50% of the cells was dose-dependently inhibited by all the factors and the strongest inhibition was demonstrated in the cells treated with TGF-beta. Although Activin A and IL-6 did not block the colony development when the agents were administered at 96 h, just before the time when the cells started to form colonies, TGF-beta and IL-1 beta could inhibit the colony formation completely and partially, respectively. Transient treatment (48-72 h) with TGF-beta was enough to suppress colony development, while Activin A and IL-6 did not block the formation of colonies. IL-1 beta partially suppressed this formation. However, continuous administration (48-144 h) of IL-beta as well as TGF-beta stimulated the detachment of the cells from dishes and the remaining hepatocytes failed to form colonies. In addition, only TGF-beta could inhibit the DNA synthesis of most small cells in the established colonies as well as that of relatively large hepatocytes. Neither Activin A, IL-1 beta nor IL-6 could inhibit the DNA synthesis of the small cells. Thus, only TGF-beta could completely inhibit both the DNA synthesis of any type of hepatocyte and the formation of small-cell colonies.

Interaction of interleukin-1 and interferon-gamma on fibroblast growth factor-induced angiogenesis.

The interaction of interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) actions on several aspects of angiogenesis in vitro and in vivo was studied. The proliferation and migration of human umbilical vein endothelial cells cultured with basic fibroblast growth factor (bFGF) were synergistically inhibited by cotreatment with IL-1 and IFN-gamma. Endothelial cell adhesion to collagen was suppressed by IL-1 and the effect was slightly enhanced by the combination of IL-1 and IFN-gamma. Local administration of IL-1 (10,000 U) and IFN-gamma (1,000 U) inhibited bFGF-induced angiogenesis in the skin of mice, and synergistic inhibitory activity of the combination was demonstrated. Expression of FGF receptors was strongly downregulated by the combination, whereas expressions of epidermal growth factor (EGF) receptors, integrin beta 1 and integrin beta 3 were not. EGF partially abrogated the growth-inhibitory effects of IL-1 and IFN-gamma. These findings indicate that IL-1 and IFN-gamma are each able to act an angiogenesis inhibitor in a situation where FGF plays a major role in angiogenesis, and the activity is synergistically enhanced when they are used in combination.

Effects of mitogens and co-mitogens on the formation of small-cell colonies in primary cultures of rat hepatocytes.

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor-alpha (TGF-alpha). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin-8 and -18. But these cells expressed neither cytokeratin-7 nor -19. When 6 x 10(5) cells were plated on 35-mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF-alpha. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF+HGF, EGF+TGF-alpha, and HGF+TGF-alpha were not different from those of the colonies induced by each mitogen alone. To examine the ability of co-mitogenic factors to induce small-cell colonies, angiotensin-II, insulin-like growth factor-I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co-mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co-mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small-cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF-alpha, or FGFs and that the co-mitogens did not influence the formation of the small-cell colonies.

Redifferentiation of proliferated rat hepatocytes cultured in L15 medium supplemented with EGF and DMSO.

Primary adult rat hepatocytes were cultured in serum-free L15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml epidermal growth factor in a 5% CO2:95% air incubator. The number of cells increased and reached about 180% of the initial value by Day 4, and after 2% dimethyl sulfoxide (DMSO) was added to the culture medium at Day 4, the cells continued to proliferate until Day 6. The number of cells reached about 210% at Day 6 and they were well maintained until Day 18. The cell number gradually decreased with time in culture, but many cells remained for more than 2 mo. On the other hand, without 2% DMSO, the cells proliferated until Day 5, but thereafter they rapidly decreased. After DMSO addition, albumin and transferrin were secreted into the medium and the production of both proteins continued for more than 2 mo. Immunocytochemically both proteins were strongly stained in the cells treated with 2% DMSO. Although the expression of G6Pase in the cells disappeared at Day 6 without DMSO, the cells treated with 2% DMSO recovered G6Pase activity at Day 16. In addition, induction of peroxisomes by 2 mM sodium clofibric acid was clearly shown in the hepatocytes at Day 14 and Day 25 using enzyme-cytochemistry. Ultrastructurally, DMSO-treated hepatocytes had many mitochondria and large peroxisomes with a crystalline nucleoid, and both gap junctions and desmosomes were well developed between the cells even at Day 40. Thus, the number of cells doubled, some differentiated functions of the primary hepatocytes were well restored by the use of 2% DMSO, and these functions were maintained for more than 2 mo.

Effect of age on the formation of small-cell colonies in cultures of primary rat hepatocytes.

The proliferation of primary cultured rat hepatocytes was observed in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml of epidermal growth factor. These proliferating cells were mainly mononucleate and formed small-cell colonies after Day 4. The small cells in focal colonies were surrounded by typical hepatocytes and were stained immunocytochemically with anti-rat albumin and anti-cytokeratin 8 antibodies. This suggests that the cells in the small-cell colonies were derived from hepatocytes. The frequency of appearance of small-cell colonies with age was examined by the use of primary cultured hepatocytes isolated from the livers of rats between the ages of 3 wk and 90 wk. In the cells from 4- to 5-wk-old rats, about 58 colonies per 1000 attached cells appeared 144 h after plating; the number of colonies rapidly decreased to about 25 in 6- to 8-wk-old rats. In adult rats, about 17 colonies were seen, and only about five colonies were observed in rats more than 80 wk old.

Inhibitory effects of cytokines on vascular endothelial cells: synergistic interactions among interferon-gamma, tumor necrosis factor-alpha, and interleukin-1.

The formation of new blood vessels is critical to tumor growth and metastasis and since endothelial cells are the principal cell type comprising such blood vessels, we carried out experiments to test whether the growth of endothelial cells in vitro can be inhibited by low doses of combinations of cytokines. We show that growth of mouse aortic endothelial cells can be inhibited in vitro by combinations of cytokines that, administered singly even at ten times higher concentrations, have only marginal effects. In particular, interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma), acting synergistically at low, subthreshold concentrations, are able to completely inhibit the growth of endothelial cells in vitro.

Interleukin 1 and tumor necrosis factor-alpha synergistically increase the production of interleukin 6 in human synovial fibroblast.

We have previously reported that synovial cells could participate in B cell differentiation processes in rheumatoid arthritis (RA) by producing interleukin-6 (IL-6) spontaneously or in response to interleukin-1 (IL-1) stimulation. In this paper, we examined the effects of tumor necrosis factor-alpha (TNF-alpha) on IL-6 production by human synovial fibroblasts. TNF-alpha, as well as IL-1, is a putative relevant molecule in the inflammatory process and in articular destruction in RA. Both IL-1 and TNF-alpha induced IL-6 production by synovial fibroblasts in a dose dependent manner. When synovial fibroblasts were stimulated by IL-1 and TNF-alpha in combination, IL-6 production increased synergistically after 48 hr of a 72 hr culture period. Kinetic studies revealed that the presence of both cytokines at the early phase of stimulation was required for the synergistic effect. These results suggest that TNF-alpha could be involved in a cytokine network in the affected joints of RA and could contribute synergistically with IL-1 to the IL-6 production by synovial fibroblasts in vivo.

[A case of systemic lupus erythematosus complicated with multifocal posterior pigment epitheliopathy].

Multifocal posterior pigment epitheliopathy (MPPE) is a rare complication of systemic lupus erythematosus (SLE) and resistant to treatment. We here report a case of SLE with MPPE, successfully treated with prednisolone (PSL) and laser photocoagulation therapy. A 38-year-old woman, who had suffered from SLE for four years, was admitted to our hospital with the complaints of left hemiparesis, right homonymous hemianopsia, and dysarthria. The findings of MRI suggested her symptoms were caused by cerebrovascular involvement of SLE. The treatment with prednisolone was started at a dose of 100 mg per day. While the dose of PSL was reduced, she began to complain of paresthesia in left face and myodesopsia of left eye. Based upon the result of fluorescein angiogram, the diagnosis of MPPE was made. Since MPPE accompanied exacerbation of neurological manifestation, we considered that MPPE was a symptom of vascular involvement of SLE. MPPE was treated with laser photocoagulation therapy and an increased dosage of PSL, with favorable results. It has not been reported, as far as we concern, that laser photocoagulation therapy is effective for MPPE caused by SLE. This case is important for the pathogenesis and therapy of MPPE with SLE.

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study.

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.

Augmentation of LDL receptor activities on lymphocytes by interleukin-2 and anti-CD3 antibody: a flow cytometric analysis.

Dormant lymphocytes are known to show little LDL receptor (LDL-R) activities. The present study was designed to determine whether or not LDL-R activities of lymphocytes from normal subjects were high enough to be measured by flow cytometry after the cells had been stimulated with recombinant interleukin-2 (IL-2) and anti-CD3 monoclonal antibody (mAb). IL-2 or anti-CD3 mAb individually provokes proliferation of lymphocytes in a serum-free medium. Proliferation rate was accelerated when the two reagents were used in combination. Stimulated cells cultured for 5 days expressed more than 85% CD3 positive, less than 0.5% CD14 positive, and less than 1.5% CD20 positive. The LDL-R activities of the cells were examined by the uptake of a fluorescence probe, DiI-labeled LDL (DiI-LDL) and analyzed by flow cytometry. Stimulated cells showed increased uptake of DiI-LDL and 84 +/- 9% were positive, whereas only 3.0 +/- 2.5% of the cells without stimulation were positive (P less than 0.001). Under the same conditions stimulated lymphocytes from a homozygous familial hypercholesterolemia (FH) patient showed little LDL-R activities; 14% of the cells were positive. Displacement assays reveal that the uptake of LDL by these cells is occurring by way of its specific pathway. These data imply the lymphocytes stimulated with the reagents used in the study might be used for detecting defects in LDL-R, perhaps defects in other genomic systems as well.

Pretreatment of human vascular smooth muscle cells with interleukin-1 enhances interleukin-6 production and cell proliferation (action of IL-1 on vascular smooth muscle cells).

Systemic vasculitis is an inflammatory disorder of blood vessels characterized by a perivascular mononuclear cell infiltration around the vessel and fibrinoid necrosis within vessel walls. Interleukin-1 (IL-1) is a multipotent inflammatory mediator and affects several properties of vascular cells. To determine whether IL-1 could contribute to the pathogenesis of vascular diseases, we examined the effect of IL-1 on B cell stimulatory factor-2/interleukin-6 (IL-6) production by cultured human vascular smooth muscle cells (SMC) and the proliferation of these cells. Supernatants of SMC stimulated IgM synthesis of human B cell line. SKW6-CL4 cells. This activity was increased (1.7 to 2.6-fold) when SMC were pretreated with IL-1 or calcium ionophore A23187 for 48 h, and was completely blocked by rabbit anti-human IL-6 antibodies. These IL-6 activities of the SMC supernatants were also assessed by using an IL-6 dependent murine hybridoma cell line. MH-60. BSF-2. In addition, we observed that pretreatment of SMC with IL-1 for 48 h stimulated growth of SMC during the 96 h incubations, as assessed by cell number (p less than 0.05). These results suggest that IL-1 may contribute to the pathogenesis of inflammatory and immunological vasculitis by the augmentation of IL-6 release and growth of SMC.

Coated fibronectin induces adhesion in the human monocytic cell line U937.

The differentiation inducing effect of coated human fibronectin (HFN) on U937 was investigated. U937 cells usually proliferate without stimuli. HFN induced the adhesion of about 20% of U937 cells in a serum free medium. HFN did not inhibit cell growth nor induce CD14 expression. The adhesion inducing activity of HFN was completely blocked by addition of anti-HFN antibody. Phorbor 12-myristate 13-acetate (PMA), another differentiation inducing agent, inhibited the growth of U937 cells but did not induce adhesion in the serum free medium. These data imply that HFN has a differentiation inducing effect on a human monocytic cell line and could be used as a biological response modifier.

Elevated plasma levels of alpha 2-plasmin inhibitor-plasmin complex in patients with rheumatic diseases. Possible role of fibrinolytic mechanism in vasculitis.

Plasma levels of alpha 2-plasmin inhibitor (alpha 2PI) and alpha 2-plasmin inhibitor-plasmin complex (alpha 2 PIPC) were measured by sandwich enzyme-linked immunosorbent assay, using a recently developed monoclonal anti-alpha 2PI antibody, in patients with collagen diseases. Twenty patients had systemic lupus erythematosus (SLE), 4 of whom also had vasculitis, 11 patients had rheumatoid arthritis (RA), 4 of whom also had vasculitis, and 5 patients had other types of vasculitis. There was no significant difference in alpha 2PI levels between the 3 patient groups and the control groups. However, plasma levels of alpha 2PIPC in all 3 patient groups were significantly higher than those in the control group. Moreover, plasma concentrations of alpha 2PIPC in SLE patients with vasculitis were statistically significantly higher than those in SLE patients without vasculitis. These concentrations were also higher in RA patients with vasculitis than in RA patients without vasculitis, although the difference was not statistically significant. Our findings indicate that measurement of plasma alpha 2PIPC levels is useful for detecting and evaluating the severity and activity of vasculitis in patients with collagen diseases.

Expression of TLiSA1 on T cells from patients with rheumatoid arthritis and systemic lupus erythematosus.

The expression of a new activation antigen, T cell lineage specific activation antigen (TLiSA1) on peripheral blood T cells from 16 rheumatoid arthritis (RA) and 8 systemic lupus erythematosus (SLE) patients and synovial fluid T cells from RA patients was determined in the context of T cell activation. The percentages of TLiSA1 positive T cells from inactive (4.6 +/- 5.2, mean +/- SE) or active RA (19.3 +/- 8.6) or inactive (1.7 +/- 2.1) or active SLE (8.7 +/- 2.7) were significantly increased compared with that of normal controls (0.7 +/- 0.4) (P less than 0.01). All patients with vasculitis showed relatively high positive percentages. The mean fluorocytometric intensity of TLiSA1 positive T cells from RA and SLE patients was significantly higher than that from normals. Percentages of TLiSA1 positive T cells from synovial fluids (21.8 +/- 4.9%) were significantly increased compared with those from peripheral blood of the same patients, indicating the local activation of T cells in patients with RA. An increase in the expression of TLiSA1 with no increase in the expression of the very late activating antigen 1 (VLA-1) was found in peripheral blood from RA, suggesting a difference in the stage of T cell activation in RA. In RA, there was a clinical correlation with levels of TLiSA1 expression on peripheral T cells. After stimulation with PHA, TLiSA1 positive percentages were increased on Day 2 and continued to increase through 5 days of culture. The maximum expression was obtained on Day 5. An increased number of TLiSA1 positive T cells belonged to OKT8. These results suggest that there is the systemic and the local activation of T cells in RA, following antigen stimulation, or a generalized nonspecific activation of immune system that could provide a means to monitor the abnormal immunologic activity in RA.

Heterogeneity of B cell responsiveness to interleukin 4, interleukin 6 and low molecular weight B cell growth factor in discrete stages of B cell activation in patients with systemic lupus erythematosus.

To investigate the differential stages of B cell activation in patients with systemic lupus erythematosus (SLE), the effects of recombinant interleukin-4 (rIL-4) interleukin-6 (rIL-6), and low mol. wt BCGF (1-BCGF) on B cell proliferation and differentiation were evaluated. In a co-stimulatory assay with anti-IgM, proliferative response to rIL-4 and 1-BCGF showed no significant differences between SLE patients and healthy controls. In a restimulation assay, after pre-activation with anti-IgM for 3 days, proliferative response to rIL-4 and 1-BCGF was significantly decreased in SLE patients compared with normal healthy controls (P less than 0.01). With regard to rIL-6 induced B cell differentiation, SAC-pre-activated low density, large B cells from both SLE patients and healthy controls differentiated well, whereas high density B cells did not differentiate at all. Of particular interest, low density B cells from active patients directly differentiated into Ig secreting cells in response to rIL-6 without SAC activation. These data indicate that heterogeneity of B cell responsiveness to B cell-tropic interleukins resulted from the discrete stages of B cell activation in SLE.

[Analysis of systemic lupus erythematosus (SLE) involving the central nervous system by magnetic resonance imaging (MRI)].

Involvement of the central nervous system (CNS) commonly occurs in systemic lupus erythematosus (SLE). But definitive diagnosis remains difficult even with computed tomography (CT). In this study, we used the recently developed technique, magnetic resonance imaging (MRI) for CNS lupus and compared it with CT scans. CT was performed with a General Electric 8800 CT/T scanner. MRI was performed using a Mitsubishi Electric MMI-150 S. Ten patients with CNS lupus were divided into 3 groups. Group I included 4 cases with neurological manifestations alone. All lesions seen on CT were also detected by MRI, with greater clarity and extent. Furthermore, MRI depicted several microinfarcts in white matter without symptoms. Group II included 5 cases with psychiatric features alone. MRI detected a thalamic microinfarct in only one case while CT showed no abnormality in all cases. Group III included 1 case with both neurological and psychiatric symptoms. MRI demonstrated a small infarct of midbrain corresponding with neurological symptoms, more clearly than CT. Therefore MRI demonstrates the degree of brain involvement in SLE more accurately than CT.

Stimulation of interleukin 6-like B-cell differentiation factor production in human adherent synovial cells by recombinant interleukin 1.

Abnormal production of immunoglobulin in the joint space is frequently observed in patients with rheumatoid arthritis (RA). We have previously demonstrated that adherent synovial cells (ASC) from patients with RA are involved in B-cell differentiation by their spontaneous production of B-cell differentiation factor (BCDF). The regulation of the production of this factor, however, has not yet been described. We investigated the effects of recombinant interleukin 1 alpha and beta (rIL-1 alpha and rIL-1 beta) on the production of BCDF in ASC. Increased production of BCDF was observed with increased rIL-1 concentration. Production of BCDF was detected 3 h after exposure of ASC to rIL-1 and increased throughout a 48-h culture. This BCDF, assayed on SKW6-CL4 cells, was found to share a common active site with interleukin 6. The effect of rIL-1 was almost neutralized by anti-IL-1 antibody and the addition of polymyxin B did not diminish the effect of rIL-1, indicating that rIL-1 itself stimulates ASC in vitro. These results suggest that IL-1 may play a regulatory role in the production of BCDF in synovial tissue.

Rheumatoid adherent synovial cells produce B cell differentiation factor activity neutralizable by antibody to B cell stimulatory factor-2/interleukin 6.

B cell differentiation factor (BCDF) activity assayed on SKW6-CL4 cells was found in adherent synovial cell (ASC) conditioned medium. ASC of patients with RA (n = 8) produced significantly larger amounts of BCDF than those of patients with osteoarthritis (n = 5) (219.0 +/- 212.1 vs 25.5 +/- 12.4 units/microgram.DNA, p less than 0.01). ASC clones, established by the limiting dilution technique, also produced this factor. Experiments with several neutralizing antibodies revealed that this BCDF is inhibited by anti-B cell stimulatory factor-2/interleukin 6 antibody (anti-BSF-2/IL-6 ab) up to 90%, but not by antiinterleukin 1, antiinterleukin 2 or antiinterferon-r antibodies. Our data suggest that ASC could participate in the B cell differentiation process in joint space by producing a molecule which has a similar active site to BSF-2/IL-6.