In vitro differentiation and characterization of human peritoneal macrophages from CAPD-peritonitis patients.

Studies on human macrophages are restricted due to difficulties in isolating significant numbers of human macrophages. High numbers of monocytes/macrophages can be obtained from peritonitis effluents of patients treated with peritoneal dialysis. To determine whether these cells might be useful for functional studies, we characterized peritoneal macrophages (PM) immediately after isolation from the dialysate effluents and their subsequent differentiation. During a 10 days culture period they differentiated morphologically and phenotypically (FACS-analysis) from monocyte-like cells to macrophages. Reflecting the intraperitoneal inflammation we found protein- and mRNA-synthesis of IL-8 and monocyte-chemoattractant-protein-1 (MCP-1) to be upregulated in PM after isolation from the effluents. In contrast, TNF-alpha was downregulated and could not be stimulated by LPS and/or IFN-gamma, reflecting the phenomenon of desensitization. After 10 days in culture, cytokine production normalized to a constitutive level and the TNF-alpha responsiveness to LPS was restored. These data suggest the recovery of PM from the inflammatory prestimulation. Therefore PM harvested from peritoneal dialysis effluents might provide a useful tool for further studies on the role of human macrophages in inflammation.

[Superoxide-dismutase and superoxide-radical-release in rheumatoid arthritis (author's transl)]. / Superoxid-Dismutase und Superoxid-Radikal-Freisetzung bei juveniler rheumatoider Arthritis.

Polymorphonuclear leukocytes (PMNs) release superoxide anion (O-2) when they are exposed to a phagocytic stimulus. Intracellularly the copper-containing enzyme superoxide dismutase (SOD) protects against the toxic effects of O-2. To investigate the role of O-2 and SOD in the inflammatory process we determined the O-2 release and SOD content in PMNs. In PMNs of children with rheumatoid arthritis the SOD activity was diminished compared to healthy controls. Upon stimulation with opsonized zymosan PMNs obtained from children with rheumatoid arthritis generated greater amounts of superoxide anion than control cells. The "SOD deficiency" in PMNs of children with rheumatoid arthritis may promote this extreme release of the toxic superoxide radical inducing the damage of the connective tissue. The involvement of superoxide dismutase and superoxide anion in inflammatory process may induce further studies, leading hopefully to an appropriate understanding or even to new principles in the treatment of the rheumatoid arthritis.

Superoxide dismutase and glutathione peroxidase in polymorphonuclear leucocytes.

Superoxide dismutase (SOD) and glutathione peroxidase (GPX) protect aerobic organisms against the toxic superoxide anion and hydrogen peroxide, which are generated during phagocytosis by polymorphonuclear leucocytes (PMNs). PMNs of children with bacterial infections and with infectious hepatitis contained significantly elevated SOD activity, whereas GPX activity remained in the normal range. In contrast, PMNs of children with viral infections and rheumatoid arthritis exhibited a decreased SOD activity, while GPX activity was again unchanged. The children's age, sex or treatment did not effect the enzyme activities in PMNs. Since SOD generates bactericidal hydrogen peroxide and regulates the release of the toxic superoxide radical into the surrounding tissues, this study may add new understanding to the pathophysiological aspects of acute and chronic inflammatory processes.

[Eruptive keratoacanthoma]. / Eruptive Keratoakanthome.

A 74 year old woman showed typical symptoms of eruptive keratoacanthomas since three years: exanthematic sudden appearance of numberless papules, few millimeters in diameter with central keratinization, masked facies, ectropion of the eyelids and pruritus. Histology revealed the characteristic architecture of keratoacanthoma. Proliferation kinetics were characterized by increase of cell division within the basal cell layer, prolongation of time for DNA-synthesis (15,7 h) and moderate decrease of generation time. The reactive inflammatory dermal infiltrate mainly consists of lymphocytes. Ultrastructural features comprise occurrence of intracellular desmosomes and elastic and collagen fibers between the tumor cells. Therapeutically a good response was observed to aromatic retinoid Ro 10-9359.

Inverse MCP-1/IL-8 ratio in effluents of CAPD patients with peritonitis and in isolated cultured human peritoneal macrophages.

An important event in intraperitoneal inflammation is the influx of leukocytes into the peritoneal cavity. Chemokines such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) play a major role in the recruitment of immune cells to the site of inflammation. We determined the concentrations of two members of the chemokine family, IL-8 and MCP-1, in the dialysate effluents of 18 continuous ambulatory peritoneal dialysis (CAPD) patients with peritonitis and of 18 non-infected CAPD patients by specific enzyme-linked immunosorbent assays (ELISA). Isolated peritoneal macrophages (PMs) from CAPD peritonitis patients were cultured and IL-8 and MCP-1 production was determined on protein (ELISA) and mRNA level (Northern blot) at designated timepoints over a 72-h culture period. PMs from non-infected patients served as controls. Much higher concentrations of IL-8 and MCP-1 were found in dialysate effluents of peritonitis patients than in effluents of non-infected patients: IL-8 2.39 +/- 1.15 vs 0.05 +/- 0.01 ng/ml and MCP-1 22.5 +/- 6.27 vs 0.42 +/- 0.07 ng/ml. IL-8 and MCP-1 release by cultured PMs from peritonitis patients and non-infected patients revealed significant differences: IL-8 40.3 +/- 2.2 ng/ml after 3 h and 194.2 +/- 34.9 ng/ml after 12 h compared to 21.02 +/- 6.15 ng/ml after 3 h and 89.64 +/- 30.28 ng/ml after 12 h, respectively; MCP-1 3.3 +/- 0.9 ng/ml after 3 h and 25.7 +/- 7.4 ng/ml after 12 h compared to 1.1 +/- 0.2 ng/ml and 1.8 +/- 0.2 ng/ml, respectively. Interestingly, the ratio of IL-8 to MCP-1 concentrations in the dialysate effluents (1:9.4) is reversed in the supernatants of cultured PMs. In the effluents and in the culture supernatants of PMs from CAPD peritonitis patients high amounts of IL-8 and MCP-1 are detectable, suggesting that PMs are an important source for these chemokines during peritonitis. Because of the inverse ratio of IL-8 and MCP-1 in the effluents and culture supernatants it can be assumed that PMs are responsible for the MCP-1 concentration to a lesser extent than for the IL-8 concentration in the effluents.

Distinct regulation of IL-8 and MCP-1 by LPS and interferon-gamma-treated human peritoneal macrophages.

BACKGROUND: Interleukin-8 and monocyte chemotactic protein-1 (MCP-1) are major leukocyte chemoattractants during bacterial peritonitis by recruiting neutrophils and monocytes/macrophages respectively. METHODS: Peritoneal macrophages (PM) from 12 different CAPD patients with peritonitis were stimulated with either 10 ng/ml LPS, 10 ng/ml IFN-gamma or LPS+IFN-gamma, and IL-8 and MCP-1 production was determined on protein and mRNA levels by using ELISA technique and Northern blot analysis. To obtain information from two different stages of activation, experiments were done with highly activated PM directly after isolation and with cells after 10 days in culture, each group being stimulated for 4 h. Unstimulated cells served as control. RESULTS: Immediately after isolation IL-8 mRNA-expression and synthesis was high and could be further increased by LPS stimulation, whereas IFN-gamma treatment showed no significant influence. The levels of MCP-1 were also initially high but could not be further stimulated by LPS, whereas addition of IFN-gamma resulted in a significant rise in MCP-1 synthesis. After 10 days in culture LPS-stimulation of cells again revealed a significant increase in IL 8 protein synthesis, whereas IFN-gamma showed no effect. LPS anergy for MCP-1 was still seen in PM after 10 days in culture, and IFN-gamma treatment again induced a significant rise in MCP-1 synthesis. The overall production of both chemokines was far higher on day 1 compared to day 10. CONCLUSION: Our data show differences in LPS/IFN-gamma regulation for IL-8 and MCP-1 in both highly activated and in resting, mature peritoneal macrophages, suggesting distinct pathways for these chemokines that may offer a means of control for the specific recruitment of neutrophils and monocytes/macrophages in bacterial peritonitis.