Inflamed synovium is characterized by high concentrations of cytokines [interleukin (IL)-6, IL-1beta and tumour necrosis factor (TNF)-alpha] and the abundant presence of infiltrated monocytes, many of which are found adjacent to the resident fibroblast-like synoviocytes. We have used a co-culture of fibroblast-like synoviocytes and differentiated U937 cells to study IL-6, IL-1beta and TNF-alpha release. After a 3 day co-culture, 35% of the U937 cells had adhered and were fully differentiated towards monocytes, as determined by expression of p47phox, CD14, MSE-1, Mac-1, collagenase and NADPH oxidase activity. IL-6 release from fibroblast-like synoviocytes was induced 4-fold by co-culture with differentiated U937 cells. However, co-culture of differentiated U937 cells with fibroblast-like synoviocytes failed to release detectable levels of IL-1beta and TNF-alpha from the U937 cells. Addition of synovial fluid further increased IL-6 release, but again had no effect on IL-1beta or TNF-alpha, although U937 cells differentiated by phorbol ester were able to release these two cytokines and, in the case of the co-culture, mRNAs for both cytokines were highly expressed in the U937 cells. We postulate that the influx of monocytes into the synovium is instrumental in the elevation of IL-6 levels, but this is not sufficient to explain high levels of IL-1beta or TNF-alpha.
Angiogenesis is the process by which new blood vessels are created from pre-existing vessels. It is essential for the growth and development of normal cells and tissues during embryonic and neonatal development and of tumour cells. Solid tumours rely on having an extensive network of blood vessels for growth and survival. The key mediator of angiogenesis, vascular endothelial growth factor-A (VEGF-A), is critical for the growth of tumours and their subsequent metastasis and is known to initiate angiogenesis. Bevacizumab is a humanized immunoglobulin G monoclonal antibody that binds to VEGF with high specificity, thereby blocking VEGF-mediated signalling pathways and thus angiogenesis. Clinical trials have shown that bevacizumab is effective in prolonging survival in patients with metastatic colorectal cancer (CRC) when combined with standard chemotherapy. Consequently, bevacizumab has been approved in combination with 5-fluorouracil-based chemotherapy for first-line treatment of patients with metastatic CRC. Bevacizumab is generally well tolerated in most patients and does not exacerbate the adverse events associated with conventional chemotherapy. Bevacizumab-related side effects are generally manageable; however, monitoring for hypertension, gastrointestinal perforation, bleeding, proteinuria and thromboembolism is advised, especially in patients with predisposing factors. In addition to demonstrated survival benefits, the convenient dosing schedule and lack of interactions should ensure the successful integration of this novel agent into clinical practice.
Rheumatoid arthritis is characterized by inflammation, hyperplasia of the synovial membrane, pannus formation and degradation of cartilage and bone. Fibroblast-like synoviocytes are thought to be involved in the invasion and subsequent degradation of cartilage. Two processes play a role in cellular invasion: cellular migration and degradation of the extracellular matrix. The adhesion molecule CD44 and chemokine receptors are instrumental in migration and invasion. Both components have been reported to play a role in tumour metastasis but also appear to be implicated in the destruction of synovial joints in rheumatoid arthritis. CD44, an ubiquitously expressed receptor for the glycosaminoglycan hyaluronan, contains 9 exons that are alternatively spliced and this gives rise to the expression of multiple splice variants, each exhibiting different functional capacities.In this report we describe an analysis of the expression of chemokine receptors and CD44 splice variants in diseased synovial tissues using the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). We have correlated our findings with the clinical diagnosis of rheumatoid or osteoarthritis, with invasion into the extracellular matrix in vitro, and with the rate of proliferation of fibroblast-like synoviocytes.We conclude that fibroblast-like synoviocytes from both osteo- and rheumatoid arthritis express a number of different chemokine receptors and CD44-splice variants, but none of these correlate with a particular diagnosis. However, elevated expression of CD44v8-9 was found to correlate negatively with the invasive capacity of fibroblast-like synoviocytes.
The neutrophil activators phorbol 12-myristate 13acetate (PMA), formyl-methionyl-leucyl-phenylalanine, serum-treated zymosan, and IgG-coated latex cause an increase in protein phosphorylation in human neutrophil cytoplasts, concomitantly with an increase in oxygen consumption.After sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography, phosphorylation was apparent in many proteins, must abundantly in 42-, 47-, 50-, 60-, and 80-kDa proteins.In neutrophil cytoplasts from autosomal chronic granulomatous disease (CGD) patients that were stimulated with PMA, the phosphorylation of a 47-kDa protein is absent.The localization of this protein in PMA-activated control cytoplasts is mainly in the cytosol and, to a lower and more variable extent, in the membrane.After addition of purified protein kinase C to lysates of nonstimulated control cytoplasts, phosphorylation occurred at the 47-kDa level in both the cytosol and the membrane fraction.With lysates of autosomal CGD cytoplasts, in vitro phosphorylation of the 47-kDa protein was completely absent.After separation of cytoplast proteins on a sodium dodecyl sulfate-polyacrylamide gel and excision of the 47-kDa protein(s), phosphorylation of the isolated 47-kDa band was observed in the presence of purified protein kinase C.This reaction was again absent when autosomal CGD cytoplasts were used as starting material.Our studies have identified the 47-kDa protein in neutrophil cytoplasts as a true substrate for protein kinase C and indicate that the defect in phosphorylation at the 47-kDa level in autosomal CGD cytoplasts is due to a defective protein.The human neutrophil shows a cyanide-insensitive respiratory burst upon phagocytic stimulation or upon exposure to soluble agonists, such as phorbol 12-myristate 13-acetate
Aim: The clinical symptoms of autoantibody (AAb)-mediated autoimmune diseases (AID) usually correlate with the AAb-titer. Immunoglobulins (Igs) of the IgG type are actively recycled by the neonatal crystallizable fragment receptor (FcRn). The most common Ig type of AAb is IgG. This explorative study evaluates the safety and tolerability of a fully human anti-FcRn monoclonal antibody (mAb) in patients with thyroid autoimmunity (TA). Methods: Adverse events (AEs) and serious AEs (SAEs) were documented and coded according to the standardized Medical Dictionary for Regulatory Activities (MedDRA). AEs were followed up, and seriousness, as defined by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)-guideline E6, was documented. All AEs were analyzed for a possible underlying cause, and if not identified, were graded as side effects (SEs). Additionally, safety-relevant serological parameters (liver function and blood cell counts) were evaluated. Furthermore, laboratory parameters influenced by other anti-FcRn agents in clinical studies were considered. Results: Of 31 patients with TA, 19 were administered the anti-FcRn mAb subcutaneously once weekly for 12 weeks, while 12 were on placebo. Compared to placebo, there was no increased occurrence of AE and/or SE in the mAb group. mAb treatment increased total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol. A mAb treatment-induced transient decrease in serum albumin strongly correlated with an increase in total cholesterol (r = –0.893, P = 0.012). Overall compared to placebo, there were no significant changes in blood cell counts, complement factors, or liver enzymes. Serological changes were transient and spontaneously normalized after treatment completion. Two SAEs were deemed no-drug induced (dysthyroid optic neuropathy and a post-COVID infection associated autoimmune encephalomyelitis). Conclusions: The anti-FcRn mAb is a safe and well-tolerated therapy for AAb-mediated AID.
The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.
To assess the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the activation of the human neutrophil respiratory burst, we have utilized an ether lipid of the type 1-0-alkyl-2-0-methylglycerol (AMG), recently shown to be an inhibitor of this kinase.AMG-C16 (with an hexadecyl chain at the sn-1 position) was found to inhibit the respiratory burst induced by suboptimal concentrations of phorbol 12,13-dibutyrate.Respiratory burst activity was recovered by subsequent addition of a supraoptimal dose of phorbol 12myristate 13-acetate, indicating that in the presence of the inhibitor only the activation of the NADPH:02 oxidoreductase via protein kinase C is inhibited, but not the oxidoreductase itself.The respiratory burst induced by the chemoattractant N-formyl-methionylleucyl-phenylalanine (fMLP) was also inhibited in the presence of AMG-C16, the extent of inhibition being dependent on the concentration of fMLP.At the concentrations applied in these studies, AMG-Cls had no effect on cell viability, did not affect the formation of inositol phosphates induced by fMLP, and did not affect the characteristics of the Ca2+ fluxes induced by the same stimulus.In a cell-free assay system, AMG-C16 had no effect on the activity of CAMP-dependent or Ca'+/calmodulin-dependent protein kinase but inhibited protein kinase C in a dose-dependent fashion.To characterize the inhibitory action of AMG-Cle on the respiratory burst activity in more detail, we studied protein phosphorylation in relation to respiratory burst activity in neutrophil cytoplasts.We focused on the phosphorylation of the 47-kDa protein, because this protein is functionally associated with the NADPH:02 oxidoreductase.At suboptimal concentrations of phorbol 12,13-dibutyrate, AMG-C16 inhibited phosphorylation of proteins, including that of the 47-kDa protein.Recovery of protein phosphorylation in parallel to recovery of respiratory burst activity was obtained by addition of increasing doses of phorbol 12,13-dibutyrate.Recovery of respiratory burst activity at intermediate concentrations of fMLP did not result in a proportional increase in 47-kDa protein phosphorylation; phosphorylation of the 47-kDa protein was recovered only at high concentrations of fMLP.From these data we conclude that protein kinase C is involved in the activation of the respiratory burst by phorbol esters and fMLP.However, with fMLP as
