Human Peritoneal Macrophages Research Articles

Uptake and intracellular activity of ketolide HMR 3647 in human phagocytic and non-phagocytic cells

To evaluate the uptake of HMR 3647 into human neutrophils (PMNs), human peritoneal macrophages (PMOs) and tissue-cultured cells (epithelial cells and fibroblasts), and to assess the intracellular activity of this drug. Cell uptake of HMR 3647 was measured by radiometric assay, as described by Klemper and Styrt. Intracellular activity was determined by incubation for 3 h of PMNs containing bacteria in the presence of HMR 3647. The intracellular concentrations were 130 and 71 times higher than extracellular concentrations in PMNs and PMOs, respectively (extracellular concentrations: 2-25 mg/L). The cellular-to-extracellular concentration ratios (C/E) for tissue-cultured cells were lower than those obtained in phagocytic cells but still greater than 5. The uptake of HMR 3647 was rapid and non-saturable in all cells. HMR 3647 was released slowly from phagocytic cells. HMR 3647 (extracellular concentration: 0.5-10 mg/L) did not significantly reduce the intracellular survival rate of Staphylococcus aureus ATCC 25923 in PMNs. HMR 3647 reaches intracellular concentrations several times higher than extracellular concentrations within phagocytic and non-phagocytic cells. The slow efflux of this drug from phagocytic cells suggests that these cells may be a vehicle for it, delivering it to sites of infection.

Regulation of TNF-α by 1α,25-dihydroxyvitamin D3 in human macrophages from CAPD patients

We have previously reported that 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] accumulates in the dialysis fluid of uremic patients treated by continuous ambulatory peritoneal dialysis (CAPD). It has been reported that this metabolite regulates the production of cytokines by monocytes/macrophages. Since tumor necrosis factor-alpha (TNF-alpha) initiates an inflammatory cascade during peritonitis, the aim of the present study was to investigate the effect of 1alpha, 25(OH)2D3 on the production of TNF-alpha by human peritoneal macrophages (HPMs). HPMs were obtained from patients on CAPD. Cells were incubated with various concentrations of 1alpha, 25(OH)2D3, 1alpha,24(S) dihydroxyvitamin D2 [1alpha,24(S)(OH)2D2] or 25-hydroxyvitamin D3 (25-OH-D3) for 16 hours. This was followed by lipopolysaccharide (LPS; 1 microg/mL) incubation for 2.5 to 6 hours. TNF-alpha protein production was determined by enzyme-linked immunosorbent assay. TNF-alpha mRNA was assayed by the reverse transcriptase-polymerase chain reaction procedure, using internal synthetic mRNA standards for quantitative results. Incubation of HPMs with 1alpha,25(OH)2D3 prior to stimulation with LPS dose dependently inhibited the expression of TNF-alpha on both mRNA and protein levels. Similar results were obtained with the less calcemic vitamin D2 analogue 1alpha,24(S)(OH)2D2. Incubation of HPMs with 25-OH-D3 also revealed a down-regulation of TNF-alpha expression. Since this down-regulatory effect was blocked by ketoconazole, it is likely that this effect was caused by the conversion of 25-OH-D3 into 1alpha,25(OH)2D3 by HPMs. 1alpha,25(OH)2D3 has a potent inhibitory effect on the production of TNF-alpha by LPS-activated HPMs. We hypothesize that 1alpha, 25(OH)2D3 may constitute a regulatory mechanism that, by controlling the intensity of the inflammatory response of the peritoneum, will moderate tissue damage during peritonitis.

"One-pot" synthesis and antimalarial activity of formamidine derivatives of 4-anilinoquinoline.

Amodiaquine (AQ) is an antimalarial which is effective against chloroquino-resistant strains of Plasmodium falciparum but whose clinical use is severely restricted because of associated hepatotoxicity and agranulocytosis. "One-pot" synthesis of formamidines likely to be transformed into AQ derivatives is reported. Compared with AQ, the new compounds were devoid of in vitro cytotoxicity upon human embryonic lung cells and mouse peritoneal macrophages. One showed a potent in vivo activity in mice infected with P berghei. Transformation of this compound by reductive amination led to a new type of AQ derivatives that displayed an in vitro activity similar to that of AQ but did not lead to toxic quinone-imines.

Specificity of a new lipid mediator produced by testicular and peritoneal macrophages on steroidogenesis.

Macrophage-derived factor (MDF) is a lipophilic factor produced by rat testicular and peritoneal macrophages that maximally stimulates testosterone production by rat Leydig cells through a steroidogenic acute regulatory protein independent mechanism. The purpose of the present study was to determine whether MDF is also produced by human macrophages, and/or if it acts on human steroidogenic cells. We also studied the tissue-specific functions of MDF by determining if it also acts on steroidogenic cells of the ovary and adrenal glands and, if so, does it require new protein synthesis. It was found that MDF was produced by human peritoneal macrophages, and was capable of stimulating human steroidogenic cells. In terms of tissue specificity, it was found that primary cultures of rat adrenocortical cells respond to MDF with increased secretion of aldosterone and corticosterone, as did rat granulosa cells by producing progesterone. MDF acted in the presence of cycloheximide, indicating that it does not require new protein synthesis. These results indicate that MDF may have significant therapeutic potential and provide a basis for future studies concerning its physiological role in humans. These results further suggest that MDF is not only involved in paracrine regulation of Leydig cells, but also has the potential for the local regulation of steroidogenesis in both granulosa and adrenal cortical cells.

Uptake and intracellular activity of ofloxacin isomers in human phagocytic and non-phagocytic cells

The penetration and intracellular activity of ofloxacin and its isomers (levofloxacin and d-ofloxacin) into human polymorphonuclear leucocytes (PMN), human peritoneal macrophages (PMφ) and tissue cultured epithelial cells (McCoy) were evaluated. The cellular to extracellular concentration (C/E) values of the three fluoroquinolones were higher than 3.6 and 2.6 in PMN and PMφ, respectively. The C/E ratios in McCoy cells were lower than those in PMN, but still higher than 2.0. The uptake of ofloxacin and its isomers was rapid, non-saturable and reversible. All quinolones (extracellular concentrations: 2, 5 and 10 mg/l) produced a significant reduction of viable intraphagocytic Staphylococcus aureus in phagocytic cells. We concluded that ofloxacin and its isomers reach high intracellular concentrations in phagocytic and non phagocytic cells while remaining active in the former.

Antineutrophil Cytoplasmic Antibodies Induce Reactive Oxygen-Dependent Dysregulation of Primed Neutrophil Apoptosis and Clearance by Macrophages

This study assessed whether anti-neutrophil cytoplasmic antibodies (ANCAs) interfere with the safe deletion of neutrophils by apoptosis and phagocytic clearance. Tumor necrosis factor (TNF)-primed neutrophils were incubated with normal IgG (N IgG) or ANCA IgG for up to 36 hours. Compared with N IgG, ANCAs accelerated constitutive apoptosis of TNF-alpha primed neutrophils, as assessed by morphology and confirmed by DNA laddering pattern on gel electrophoresis, and accelerated progression to secondary necrosis. The accelerated apoptosis induced by ANCA was dependent on reactive oxygen species generation, as primed neutrophils from patients with chronic granulomatous disease failed to show an effect of ANCAs on apoptosis. However, there was no change in the rate at which neutrophils exhibited annexin V binding, indicating that externalization of phosphatidylserine was not accelerated by ANCAs. Furthermore, when ANCA-treated primed neutrophils were interacted with human or murine peritoneal macrophages after 12 hours there was significantly less phagocytosis by human macrophages and no difference in phagocytosis by murine peritoneal-derived macrophages when compared with N IgG-treated controls. In conclusion, ANCAs accelerate apoptosis and secondary necrosis in TNF-primed neutrophils by a mechanism dependent on the generation of reactive oxygen species, with uncoupling of nuclear and surface membrane changes, resulting in a "reduced window of opportunity" for phagocytic recognition and engulfment before disintegration.

Clearance of cationized LDL cholesterol from a muscle depot is not enhanced in human apolipoprotein A-IV transgenic mice.

Human apolipoprotein A-IV (apoA-IV) transgenic mice fed an atherogenic diet were shown previously to develop less atherosclerosis than control mice. The question arose whether the antiatherogenic effect of human apoA-IV is due to enhancement of reverse cholesterol transport despite no increase in plasma high-density lipoprotein (HDL) cholesterol. We studied male and female mice overexpressing human apoA-IV and their wild-type (WT) controls, all of which were fed a chow diet. Plasma total and HDL cholesterol and total phospholipids were not increased in the transgenic mice, and regression analysis showed no correlation between plasma levels of cholesterol or phospholipids and plasma human apoA-IV. To study reverse cholesterol transport in vivo, the disappearance of cholesterol from a depot of [(3)H]cholesterol-labeled cationized low-density lipoprotein injected into the rectus femoris muscle was compared in high expressers of human apoA-IV and WT controls. The loss of radioactivity and the diminution of the exogenous cholesterol mass were determined on days 8 and 12 after injection. No enhanced loss of radioactivity or cholesterol mass was seen in the transgenic mice even at levels of 2500 mg/dL of human apoA-IV. In some instances, there was even slower loss of exogenous cholesterol (radioactivity and mass) in the transgenic mice. Although [(3)H]cholesterol efflux from cultured human skin fibroblasts and mouse peritoneal macrophages was only approximately 30% higher in the presence of sera from high expressers of human apoA-IV, addition of phosphatidylcholine liposomes enhanced the efflux in both groups to the same extent. Another paradoxical finding was that the cholesterol esterification rate in plasma was 34% to 36% lower in human apoA-IV mice than in WT controls. In conclusion, even though apoA-IV was found previously to be atheroprotective under hypercholesterolemic conditions, high plasma levels of human apoA-IV did not enhance cholesterol mobilization in vivo in normocholesterolemic mice.

IL-2 Enhances Standard IFNγ/LPS Activation of Macrophage Cytotoxicity to Human Ovarian Carcinoma in Vitro: A Potential for Adoptive Cellular Immunotherapy

Objective. The objective was to evaluate the enhancement of human peritoneal macrophage cytotoxic in vitro activity by the addition of interleukin-2 (IL-2) to the standard interferon gama (IFNγ) and lipopolysaccharide (LPS) activation procedure used for cellular adoptive immunotherapy in a human ovarian cancer system. This cytotoxic effect of these activated macrophages was tested on cells from ovarian cancers of various stages, histology type, and grade, both prior to chemotherapy and at recurrence, in ovarian carcinoma cells lines and normal cells. Increased activation of the macrophage may make it a better candidate for intraperitoneal cellular adoptive immunotherapy as a component of ovarian cancer therapy. This was not a study of the mechanism of macrophage killing.Methods. Ascites specimens were collected from 24 ovarian cancer patients at the time of surgery or by paracentesis. The mononuclear cell fraction was isolated by discontinuous density gradient centrifugation and used as a cellular source of peritoneal macrophages (PMs) and primary cultured ovarian cancer cells. PMs were separated by 1-h adhesion followed by intensive washing to remove floating cells. The floating cells were cultured for 24 h which left the cancer cells attached after unattached cells were removed by washing. These cells formed a monolayer of cancer cells, which could be subcultured in 22 patients. The cells from the third to fifth passages were used as target cells without coculture with other cells. PMs were identified by latex ingestion, and their purity after isolation by adhesion culture was tested by flow cytometry and immunofluorescence. PMs were activated by culturing in the presence of IFNγ, with or without IL-2, for 18 h followed by the addition of LPS 6 h prior to use as effector cells in cytotoxicity assays. Ovarian cancer cells of both established cell lines and primary cultures were labeled with 51Cr and utilized as target cells to quantitatively measure PM-mediated cytotoxicity. Ovarian cancer cells were also cocultured with PMs for morphologic observations to provide supporting evidence to the cytotoxicity assays.Results. IL-2 enhances the cytotoxicity of the standard IFNγ/LPS macrophage activation in this system. Peritoneal macrophages so activated are cytotoxic to autologous and allogenic primary cultured ovarian tumors and to ovarian carcinoma cell lines. The macrophages are cytotoxic to cells both prior to treatment and at recurrence, but the data from the few recurrent patients did reach statistical significance. This cytotoxicity is not MHC associated. Normal cells are minimally affected.Conclusions. IL-2 augmented the standard IFNγ/LPS method of activating peritoneal macrophage cell killing of human ovarian cancer cells in this in vitro system. The cell killing occurred with autologous and allogenic tumor cells from patients with primary and possibly recurrent tumors. Activated PMs minimally affected the normal cells tested. This enhanced activation may improve the disappointing results of previous adoptive cellular immunotherapy human trials and should be considered for ovarian cancer clinical trials.

Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro.

Oxidized LDL is implicated in atherosclerosis; however, the pathways that convert LDL into an atherogenic form in vivo are not established. Production of reactive nitrogen species may be one important pathway, since LDL recovered from human atherosclerotic aorta is enriched in nitrotyrosine. We now report that reactive nitrogen species generated by the MPO-H2O2-NO2- system of monocytes convert LDL into a form (NO2-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation, essential steps in lesion development. Incubation of LDL with isolated MPO, an H2O2-generating system, and nitrite (NO2-)-- a major end-product of NO metabolism--resulted in nitration of apolipoprotein B 100 tyrosyl residues and initiation of LDL lipid peroxidation. The time course of LDL protein nitration and lipid peroxidation paralleled the acquisition of high-affinity, concentration-dependent, and saturable binding of NO2-LDL to human monocyte-derived macrophages and mouse peritoneal macrophages. LDL modification and conversion into a high-uptake form occurred in the absence of free metal ions, required NO2-, occurred at physiological levels of Cl-, and was inhibited by heme poisons, catalase, and BHT. Macrophage binding of NO2-LDL was specific and mediated by neither the LDL receptor nor the scavenger receptor class A type I. Exposure of macrophages to NO2-LDL promoted cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation. Collectively, these results identify MPO-generated reactive nitrogen species as a physiologically plausible pathway for converting LDL into an atherogenic form.

Nitric Oxide Production in Peritoneal Macrophages from Peritoneal Dialysis Patients with Bacterial Peritonitis

Nitric oxide (NO) is produced by various cell types, and it is an important mediator in many biological processes, including macrophage-mediated cellular host defense. The relevance and amount of NO production in peritonitis during peritoneal dialysis (PD) treatment is still not clear. We studied whether human peritoneal macrophages (PMphi) isolated from healthy PD patients or PD patients with peritonitis showed different spontaneous or lipopolysaccharide (LPS)/interferon gamma (IFN-gamma)-induced NO production (LPS, 1 ng/mL-10 microg/mL; IFN-gamma, 10-1000 U/mL; incubation between 6-48 hours; measured by Griess reagent). Results were compared with human blood monocytes (HBM) isolated from buffy coats. Inducible nitric oxide synthetase (iNOS) mRNA expression was looked for in PMphi by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, plasma (P) and peritoneal dialysate effluent (D) nitrite concentrations were measured in vivo. The dialysate-to-plasma ratio (D/P) of nitrite concentration was inverse in the case of peritonitis compared to infection-free patients (peritonitis D/P = 1.3, non peritonitis D/P = 0.4; p < 0.01). PMphi from peritonitis patients produced higher amounts of NO than did those from infection-free patients (0.040+/-0.044 nmol per microgram cell protein versus 0.018+/-0.015 nmol per microgram cell protein, p < 0.05). NO release could not be further enhanced by stimulation with LPS plus IFN-gamma (1 ng/mL, 250 U/mL, respectively). However, NO production in PMphi from infection-free patients increased during in vitro stimulation (0.044+/-0.031 nmol per microgram cell protein versus 0.018+/-0.015 nmol per microgram cell protein, p < 0.01). An increase of iNOS mRNA expression could be demonstrated by RT-PCR. Blood monocytes from healthy donors also increased NO release during cytokine stimulation (0.032+/-0.015 nmol per microgram cell protein versus 0.019+/-0.009 nmol per microgram cell protein, p < 0.05). Our results indicate that significant amounts of NO are released intraperitoneally in the case of bacterial peritonitis. PMphi represent a site of NO production, though the absolute amounts released in vitro are only moderate. NO production can be induced in PMphi and HBM by LPS/IFN-gamma stimulation in vitro.

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-α was downregulated and could not be stimulated by LPS and/or IFN-γ, reflecting the phenomenon of desensitization. After 10 days in culture, cytokine production normalized to a constitutive level and the TNF-α 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.

The effect of a high partial pressure of carbon dioxide environment on metabolism and immune functions of human peritoneal cells—Relevance to carbon dioxide pneumoperitoneum

Objective: Our purpose was to evaluate in vitro the effect of a high partial pressure of carbon dioxide environment used in laparoscopy on metabolic and immune response of various human peritoneal cells. Study Design: Polymorphonuclear leukocytes were obtained from 5 healthy volunteers, peritoneal macrophages were obtained from the effluent of 8 patients undergoing continuous ambulatory peritoneal dialysis, and human peritoneal mesothelial cell cultures were prepared from omentum derived from 5 patients undergoing elective surgery. The cells were exposed to a laparoscopy-like environment (1 atmosphere carbon dioxide and 0.2 atmosphere oxygen), to a control gas mixture (1 atmosphere helium and 0.2 atmosphere oxygen), or air for 3 hours. After exposure to gas mixtures, cell functions were tested at various recovery periods. Results: Three hours of exposure to a high partial pressure of carbon dioxide had no effect on viability of peritoneal macrophages and human peritoneal mesothelial cells, tested by trypan blue dye uptake and lactate dehydrogenase release. A high partial pressure of carbon dioxide decreased the mitochondrial dehydrogenases activity of peritoneal macrophages and human peritoneal macrophage cells by 60%, assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. High partial pressure of carbon dioxide blocked the superoxide release from activated polymorphonuclear leukocytes and the secretion of interleukin 1β from stimulated peritoneal macrophages, and human peritoneal macrophage cells were decreased by 15% and 30% and the secretion of tumor necrosis factor-α from peritoneal macrophages was suppressed by 85%. Mitochondrial activity, polymorphonuclear leukocyte function, and interleukin 1β and tumor necrosis factor-α secretion returned to normal after a recovery period of 12 to 24 hours, 4.5 hours, and 24 hours, respectively. In the control experiments exposure of cells to helium had no suppressive effect. Conclusions: Exposure of cells to a high partial pressure of carbon dioxide environment suppresses the inflammatory and metabolic responses of peritoneal cells. We suggest that this suppressive effect may contribute to the low postsurgery adhesion formation and the reduction in postoperative pain observed in laparoscopy. Nevertheless, the suppression of the immune response should also be taken into account for operations involving a high risk of bacterial dissemination. (Am J Obstet Gynecol 1998;179:1503-10.)

Human osteoclast formation from blood monocytes, peritoneal macrophages, and bone marrow cells.

Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)2D3 and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)2D3, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS.

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

Interleukin-8 and monocyte chemotactic protein-1 (MCP-1) are major leukocyte chemoattractants during bacterial peritonitis by recruiting neutrophils and monocytes/macrophages respectively. 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. 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. 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.

Human peritoneal monocytic cells: Lipoprotein uptake and foam cell formation

Human peritoneal cells isolated from dialysis effluent have in vivo maturated human macrophages that could serve as a model for studying lipoprotein metabolism and foam cell formation. We previously characterized the low density lipoprotein (LDL) and acetylated LDL (acetyl-LDL) receptor activities of human total peritoneal cells. Now, we provide evidence that both LDL and acetyl-LDL stimulate acylCoA cholesterol:acyl transferase (ACAT) activity of peritoneal cells. Prolonged incubation of cells with LDL results in suppression of ACAT activity, while incubation with acetyl-LDL results in elevated and sustained enzyme activity. When human peritoneal cells were analyzed using flow cytometry, the cell population showed reactivity for CD2, CD4, CD8, CD20, CD14 and HLA-DR antigens. Purified human peritoneal mononuclear cells degraded LDL. Human peritoneal macrophages formed foam cells when exposed to LDL or acetyl-LDL in culture, and lipid deposition increased with incubation time. Macrophages incubated in the presence of butylated hydroxy toluene and LDL did not form foam cells.

Human endothelial culture supernatant selectively promotes IgM-producing hybridomas

We compared the effect of human endothelial cell culture supernatant (HECS), ESG (Ewing sarcoma growth factor), IL-6 (interleukin-6) and peritoneal macrophages on the recovery of hybridoma cells after fusion with respect to growth, stability and distribution of isotype variants. A selective growth of murine IgM-producing hybridoma cells was observed in the presence of HECS after cell fusion.

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.

Potent inhibition of HIV type 1 infection of mononuclear phagocytes by synthetic peptide analogs of HIV type 1 protease substrates.

The HIV-1 genome encodes a protease that is required for viral processing of the precursor polyproteins Pr55gag and Pr160gag-pol. Interference with this process in human lymphocytes inhibits production of infectious virus. We tested the ability of several protease inhibitors to decrease replication of HIV-1BaL in human monocytes and peritoneal macrophages. The compounds tested are oligopeptide analogs of HIV-1 protease substrates in which the scissile dipeptide has been replaced by a hydroxyethylene isostere. The protease inhibitors were added only once, 1 hr prior to inoculation with virus. Every 3-5 days, half the medium was replaced with fresh medium. Inhibition of virus production was assessed by measuring reverse transcriptase (RT) activity in supernatant medium 14 days after infection. The concentration of drug required to inhibit infection by 50% (IC50) in monocytes ranged from 0.17 to 2.99 microM; IC50 values for peritoneal macrophages ranged from 0.21 to 1.9 microM. The IC50 values for these compounds were 1.1- to 10-fold higher when tested in monocytes compared to their inhibitory effect in lymphocytes, although still potently effective in the dosage range that appeared nontoxic to cells. Cell toxicity was seen only at concentrations greater than 10 microM, and varied among the drugs tested. Immunoblot analysis of two of the drugs (SB205700 and SB108922) confirmed inhibition of polyprotein processing. In control cells, 22% of viral protein pr55 was processed to p24 by 24 hr, and 51% was processed by 48 hr. In cells treated with the protease inhibitors (2 microM), Pr55 processing was inhibited 77% at 24 hr and 89% at 48 hr. Thus, these synthetic peptide analogs potently inhibit productive infection of mononuclear phagocytes by HIV-1. Drugs of this class may be useful for the treatment of HIV-1 infection in humans.

Synergistic and selective stimulation of gelatinase B production in macrophages by lipopolysaccharide, trans-retinoic acid and CGP 41251, a protein kinase C regulator.

The production of gelatinase B by macrophages is relevant in the immunological and migratory functions of macrophages. CGP 41251, an inhibitor of protein kinase C (PKC), was found to stimulate the expression of gelatinase B in macrophages, as shown by the study of two different monocytic/macrophagic cell lines, mouse RAW 264.7 and human THP-1 cells. When human monocytes and rat peritoneal macrophages were treated with CGP 41251, insignificant increases of 10 and 25% were obtained. This can possibly be due to the presence of contaminating cells in these two enriched populations, since the CGP 41251 treatment of non-macrophagic cell lines inhibited their PMA-induced gelatinase B production. Taken together, these results suggest that the stimulatory effect of CGP 41251 is specific to cells of the monocytic lineage. Using RAW 264.7 cells as a model, the effect of CGP 41251 is additive to that obtained using lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), as revealed by gelatin zymography and Northern blot analysis. The stimulatory effect of CGP 41251 on gelatinase B production in RAW 264.7 was: (a) inhibited by calphostin C (as is the LPS-induced response), indicating a PKC-dependence; (b) inhibited by dexamethasone (as opposed to the LPS-induced response); and (c) enhanced by addition of trans-retinoic acid (RA). In fact, RA can induce gelatinase B production, either alone or in synergy with LPS and/or CGP 41251, since the combination of the three agents gives the highest gelatinase B response, at both the protein and the mRNA levels. This represents an important observation considering the RA is now being tested as an anti-cancer agent and proposed for prevention studies.

6.10#119 - HLA-DPB gene expression in human peritoneal macrophages is upregul ted by trauma

6.10#119 - HLA-DPB gene expression in human peritoneal macrophages is upregul ted by trauma