Abstract 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 (2.5×10 6 cells) and umbilical vein endothelial cells (HUVECs; 5.0×10 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-1β (IL-1β)–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)-1α/β and anti–tumor necrosis factor-α 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. Immunohistochemistry revealed that monocytes adherent to or having migrated across unstimulated HUVEC monolayers as well as the HUVECs themselves expressed MCP-1 protein. However, nonadherent monocytes failed to express it. This finding suggests that the monocyte–endothelial cell adhesive interaction results in an MCP-1–inductive signal to each cell type. MCP-1 expression by migrated monocytes may indicate that monocytes are primed to produce MCP-1 during transmigration and can secrete it in normal tissue in which inflammatory cytokines that induce MCP-1 are otherwise absent.
Human alpha(1)-microglobulin was isolated from the urine of patients with tubular proteinuria, and its molecular weight was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 33,000 daltons. The carbohydrate content was 21.7%. Anti-alpha(1)-microglobulin serum was prepared and observed to react monospecifically in gel diffusion to purified alpha(1)-microglobulin, as well as to normal human serum and urine. Sera from the domestic chicken, mouse, rat, rabbit, dog, calf, cow, goat, sheep, and horse, however, did not react to anti-alpha(1)-microglobulin serum in immunodiffusion. The lymphocyte culture supernate was found to contain alpha(1)-microglobulin. Both thymus-derived(T)- and bone marrow-derived(B)-lymphocyte culture media clearly displayed a specific precipitin line against anti-alpha(1)-microglobulin serum when tested with the Ouchterlony immunodiffusion method. The tissue distribution of alpha(1)-microglobulin was studied under immunofluorescence, and a positive staining was recognized on the lymphocyte surface. Identical staining patterns were noted on both T and B lymphocytes, though B lymphocytes took a more intense stain. It would thus seem quite possible that lymphocytes are the primary source of alpha(1)-microglobulin and that this is filtered through the glomerular basement membrane and partly reabsorbed by the renal tubules. This, then, would suggest the possibility that alpha(1)-microglobulin shares some immunological role in vivo with lymphocytes and(or) is one of the membrane proteins of lymphocytes.
Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.
AIMS--To evaluate the influence of interleukin-8 (IL-8) and other inflammatory cytokines (IL-6, IL-1 beta and tumour necrosis factor alpha (TNF alpha)) on the occurrence of peritonitis in patients receiving continuous ambulatory peritoneal dialysis (CAPD). METHODS--The study population comprised 12 patients with peritonitis, 33 without peritonitis, all undergoing CAPD, and five patients undergoing peritoneal catheter implantation. Cytokine concentrations in dialysis fluid were determined by immunoassay and their values compared. RESULTS--Concentrations of both IL-8 (median 147 pg/ml, range 20-2273 pg/ml; n = 12) and IL-6 (median 1120 pg/ml, range 96-10,600 pg/ml) were substantially elevated, while the IL-1 beta concentration was lower and TNF alpha was not detectable in patients at diagnosis. The IL-6 concentration was also elevated in patients undergoing catheter implantation as well as in those with peritonitis. The IL-8 concentration, however, was elevated only upon infection. Intraperitoneal production of IL-8 was evident on determination of paired serum and dialysis fluid cytokine concentrations, and immunostaining of peritoneal cells with monoclonal anti-IL-8 antibody. CONCLUSIONS--These results suggest that determination of the IL-8 concentration in dialysis fluid maybe useful as a specific marker for following patients with peritonitis receiving CAPD.
Small ubiquitin‐related modifier (SUMO) modification appears to regulate the activity, intracellular localization, and stability of the targeted proteins. To explore the relationship among sumoylation, antitumor reagent, and apoptosis, we treated green fluorescence protein (GFP)‐SUMO‐1‐overexpressed K562 cells (K562/GFP‐SUMO‐1) with mitoxantrone (MIT) as an antitumor reagent. By the treatment with MIT, GFP‐SUMO‐1 formed foci in nuclei. While by the treatment with a tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), GFP‐SUMO‐1 located homogeneously in nuclei. When K562/GFP‐SUMO‐1 cells were treated with TPA plus MIT, GFP‐SUMO‐1 foci became larger and apoptosis was induced more than with MIT alone. The apoptosis induced by TPA plus MIT was prevented by blockage of GFP‐SUMO‐1 foci by small interfering RNA (siRNA) against SUMO‐1. The formation of GFP‐SUMO‐1 foci was reduced by a MEK inhibitor U0126 or a nuclear export inhibitor leptomycin B, and endogenous SUMO‐1 foci were reduced in K562 cells expressing the dominant‐negative MEK1 mutant. These results suggest that the formation of SUMO‐1 foci is regulated by the MEK‐ERK pathway and may induce apoptosis. ( Cancer Sci 2007; 98: 569–576)
Interleukin 8 (IL-8) is the inflammatory cytokine that activates neutrophils. Recently, it has been reported activated neutrophils contribute to the cartilage degradation. Previous reports have been suggested interleukin-8 (IL-8) exists in high concentration in RA synovial fluid. In vitro study, various cells such as synoviocytes, monocytes/macrophages, fibroblasts and endothelial cells were reported to produce IL-8. We tried to detect IL-8 producing cells on RA synovial tissues by immunohisto-chemistry and in situ hybridization. In RA synovial tissues, IL-8 products and mRNA were detected on synovial lining cells, fibroblastoid cells, monocytes/macrophages, endothelial cells, and neutrophils. Intensity of IL-8 expression was correlated with the inflammation of synovial tissues.On the other hand, IL-8 scarecely expressed on synovial lining cells and endothelial cells in osteoarthritic synovium. These data suggest IL-8 may play an important role on inflammation and joint destruction in RA synovial tissues.
Interleukin-12 (IL-12) is a heterodimeric cytokine comprising p40 and p35 subunits produced mainly by monocytes and macrophages, and plays an essential role in the regulation of the differentiation of Th1 cells. Green tea polyphenols exhibit potent anti-oxidative activities and anti-inflammatory effects by modulating cytokine production. We investigated the effect of catechins on IL-12p40 production in murine macrophages induced by bacterial lipopolysaccharide (LPS). Pretreatment with several catechins at doses of 0.3-30μM suppressed IL-12 p40 production by murine peritoneal exudate cells (PEC) and 1774.1 cells in a dose-dependent manner. Decreases in protein production were primarily due to down-regulation of the transcription of IL-12p40 mRNA. Of the various catechins, (-)-epigallocatechin gallate (EGCG) was the most potent inhibitor, followed by (-)-gallocatechin gallate (GCG) and (-)-epicatechin gallate (ECG). EGCG inhibited LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase (JNK), while EGCG augmented LPS-induced phosphorylation of p44/p42 extracellular signal-related kinase (ERK). In addition, both EGCG and GCG inhibited LPS-induced degradation of IκBα with concomitant inhibition of nuclear protein binding to NF-κB site and synthesis of IRF-1. These results suggest that gallate-containing catechins, particularly EGCG, inhibits LPS-induced IL-12p40 production in murine macrophages by inhibiting p38 MAPK while enhancing p44/p42 ERK, leading to the inhibition of 1κs xdegradation and NF-κB activation.
