Analysis of monocyte chemotactic protein-1 production in different major histocompatability complex-restricted antigen presentation systems.

In the present study the production of the CC chemokine monocyte chemotactic protein-1 (MCP-1) in several MHC II-restricted antigen presentation systems was investigated in vitro. To assess which type of antigen-presenting cell (APC) influences MCP-1 production during antigen presentation, cultures enriched for different APC populations were prepared and MCP-1 production was determined. Our results showed that APCs that effectively induce a T cell response also produce elevated amounts of MCP-1. The MCP-1 production is highest in the memory-driven secondary response against a single antigen. Despite a massive T cell proliferation, low MCP-1 concentrations are found in Con A-induced cultures. These results suggest that T cell proliferation alone is not sufficient for MCP-1 production and that stimulation of the APC during the process of antigen presentation results in MCP-1 production. Based on our results and the literature, we propose a model for MCP-1 as an enhancer of the adaptive immune response.

Relation between responses in the neutral red retention test and the comet assay and life history parameters of Daphnia magna.

Responses of the neutral red retention (NRR) assay as test for lysosomal stability and the comet assay as test for DNA integrity were measured in the water flea, Daphnia magna, and compared with mortality and effects on population growth rate during short- or long-term exposure to seven different toxicants. The NRR test and the comet assay were performed with fresh preparations of pieces of tissue from the digestive tract or with cell preparations from whole daphnias. Five toxicants caused responses of the NRR test or the comet assay after short-term exposure at concentrations below the acute toxicity level. Preliminary results of long-term exposure experiments suggest that these biomarker responses can be related to chronic effects on survival and/or reproduction of D. magna. This type of research should provide the basis for future use of the NRR test and the comet assay as early warning biomarkers for effects of toxicants on Daphnia populations.

Infection of human endothelial cells with Staphylococcus aureus induces the production of monocyte chemotactic protein-1 (MCP-1) and monocyte chemotaxis.

Bacterial infection coincides with migration of leucocytes from the circulation into the bacterium-infected tissue. Recently, we have shown that endothelial cells, upon binding and ingestion of Staphylococcus aureus, exhibit proinflammatory properties including procoagulant activity and increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression on the cell surface, resulting in hyperadhesiveness, mainly for monocytes. The enhanced extravasation of monocytes to bacterium-infected sites is facilitated by the local production of chemotactic factors. From another study we concluded that the locally produced chemokine MCP-1 is important in the recruitment of monocytes to the peritoneal cavity in a model of bacterial peritonitis. In the present study we investigated whether cultured human endothelial cells after infection with bacteria produce and release MCP-1, which in turn stimulates monocyte chemotaxis. We observed that endothelial cells released significant amounts of MCP-1 within 48 h after ingestion of S. aureus. This was dependent on the number and the virulence of the bacteria used to infect the endothelial cells. The kinetics as well as the amount of MCP-1 released by S. aureus-infected endothelial cells differed markedly from that released by endothelial cells upon stimulation with IL-1beta. Supernatant from S. aureus-infected or IL-1beta-stimulated cells promoted monocyte chemotaxis which was almost entirely abrogated in the presence of neutralizing anti-MCP-1 antibody, indicating that most of the chemotactic activity was due to the release of MCP-1 into the supernatant. Our findings support the notion that endothelial cells can actively initiate and sustain an inflammatory response after an encounter with pathogenic microorganisms, without the intervention of macrophage-derived proinflammatory cytokines.

Chemokines produced by mesothelial cells: huGRO-alpha, IP-10, MCP-1 and RANTES.

Recently we showed the in vivo relevance of chemokines in cases of bacterial peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients. Mesothelial cells, the most numerous cells in the peritoneal cavity, are hypothesized to function as a main source of chemokine production. We investigated the time- and dose-dependent expression patterns of four chemokines by mesothelial cells at the mRNA and protein level in response to stimulation with physiological doses of proinflammatory mediators that are present at the site of bacterial inflammation. Besides the chemokines huGRO-alpha (attractant for neutrophils), MCP-1 and RANTES (monocyte attractants), the expression and production of IP-10 was analysed. Mesothelial cells were cultured and stimulated with either IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or IFN-gamma or combinations of these. The time- and dose-dependent mRNA expression of the chemokines was determined by Northern blot analysis and the protein production by ELISA. It was concluded that mesothelial cells could indeed be triggered by the mentioned stimuli to induce mRNA and protein production (huGRO-alpha and IP-10) or to augment constitutive protein production (MCP-1). However, RANTES mRNA and protein production could only be induced in some cases and only in small amounts. The chemokine response of mesothelial cells was regulated differentially, depending on the stimulus and the chemokine measured. In distinct cases, combination of the stimuli led to synergy in mRNA expression and protein production. The presented in vitro data support our hypothesis that mesothelial cells in vivo are the main source of relevant chemokines in response to proinflammatory mediators, suggesting an important role for mesothelial cells in host defence.

Identification of the major chemokines that regulate cell influxes in peritoneal dialysis patients.

To investigate which members of the recently discovered family of chemotactic cytokines (chemokines) are important in leukocyte recruitment to a bacterial inflammation site, four different chemokines in the effluent of peritoneal dialysis patients suffering from acute bacterial peritonitis were measured. The presence of two neutrophil-attracting chemokines, interleukin-8 and human melanoma growth-stimulating activity (huGRO alpha), and two monocyte-attracting members of the chemokine superfamily, monocyte chemotactic protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES), was investigated in patient effluents just before, during, and after a peritonitis episode. This was studied in seven peritonitis effluents of five patients by using chemokine-specific enzyme-linked immunosorbent assays. Cell populations in the dialysates were differentiated on cytocentrifuge preparations. The contribution of the detected chemokines to neutrophilic and monocytic cell influxes in the inflamed peritoneal cavity was analyzed by correlating concentrations of chemokines to the relevant cell numbers present in the dialysates of these patients. The detection of the neutrophil-attracting chemokine interleukin-8 during peritonitis was in accordance with other studies. Moreover, a second neutrophil chemoattractant, huGRO alpha, was identified in vivo. Both were elevated during inflammation (P < 0.02) and contributed significantly to the neutrophilic cell influx (P < 0.05). One of the monocyte-attracting chemokines, RANTES, could not be detected in any of the effluents, whereas the other, MCP-1, was significantly elevated during peritonitis (P < 0.02). In contrast to the other chemokines measured, MCP-1 concentration was relatively high in steady-state peritoneal dialysates. An absolute correlation between dialysate MCP-1 concentration and the number of macrophages in these effluents was absent. However, in a 48-well chemotaxis assay, monocyte migration toward peritonitis, as well as steady-state patient dialysates, could be blocked with antibodies to MCP-1. It was concluded, therefore, that MCP-1 is the most important monocyte chemoattractant in peritoneal dialysis steady-state and peritonitis patients; whereas, besides interleukin-8, huGRO alpha was identified as a major neutrophil-attracting chemokine in the peritonitis situation.

Detection of CD 14 on migrated monocytes by specific antibody: a possible quantification for blood monocyte chemotaxis.

In the present study we investigated the possibility to use antigen-antibody recognition for detection of monocyte chemotaxis in the 48-well microchamber assay. The described method is based on recognition of cell-specific antigenic determinants present on the migrated monocytes. After conventional 48-well chemotaxis, the migrated cells were incubated with an antibody against the monocyte surface marker CD14 (3C10 hybridoma). Subsequent incubation with enzyme-coupled antibodies and their substrate allowed the antigen and hence the migrated cells carrying this antigen, to be detected and measured in a microplate reader. Our results show that chemotaxis of normal blood monocytes towards the monocyte chemoattractants FMLP and MCP-1 could be detected with the anti-CD14 antibody 3C10 in combination with a horse-radish peroxidase coupled antibody, and that the optical density is a measure for cell number per well (positive correlation, r = 0.95). Incubation of monocytes with the applied chemoattractants FMLP and MCP-1 did not change the CD14 expression as was determined by FACScan analysis. Therefore we conclude that it is possible to use antibodies directed against antigenic determinants like CD14 to detect blood monocyte migration in a more objective way compared to subjective counting of cells on a filter. Eventually, this method can be valuable, especially for chemokine research since chemokines exert their effects on specific target cell populations. By varying the detection antibody, other cell populations besides monocytes may be quantified.

Intraperitoneal interleukin-8 and neutrophil influx in the initial phase of a CAPD peritonitis.

OBJECTIVE: To investigate whether or not a change in dialysate interleukin-8 (IL-8) concentration precedes the onset of clinically overt peritonitis and is significant in the recruitment of granulocytes during continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis. DESIGN: CAPD patients stored their overnight effluent at 4 degrees C, which was routinely thrown away after 2 days. If peritonitis developed, patients delivered their effluent of the preceding two nights and the peritonitis effluent for analysis. A control study was performed 1 to 3 months after recovery. Dialysate samples were analyzed for number of cells, differential cell count, IL-8 and elastase concentrations, and their neutrophil chemoattractive capacity. In addition, serum samples during peritonitis were analyzed for IL-8 concentrations. RESULTS: Ten peritonitis episodes in 7 patients were analyzed. Numbers of neutrophils and levels of dialysate IL-8 and elastase started to increase 4 to 12 hours before the first peritonitis effluent. The dialysate/serum IL-8 ratio was 423.5 during peritonitis and 7.0 in the postperitonitis controls. There was a significant correlation between the number of neutrophils and IL-8 concentration in the dialysate. The in vitro neutrophil chemotaxis was increased toward the peritonitis effluents, as compared to control effluents. Incubation of the peritonitis effluents with anti-IL-8 monoclonal antibody blocked the increase in neutrophil chemotaxis above control levels by an average of 26.7%. CONCLUSION: IL-8 is produced in the peritoneal cavity during CAPD treatment and may mediate part of the neutrophil recruitment and degranulation in the initial phase of a CAPD peritonitis.

Analysis of the peritoneal cellular immune system during CAPD shortly before a clinical peritonitis.

We analysed the peritoneal cellular immune system 24-48 h before the onset of a clinical peritonitis. Peritoneal cells were obtained from the overnight dialysis effluents 1 or 2 days (day-1 and day-2) preceding the day of peritonitis, the last overnight effluent before the peritonitis effluent (day P), and the first peritonitis effluent. Nine peritonitis episodes of six patients were studied. The number of Fc receptor positive cells, the chemotactic activity, and immunophenotype of the peritoneal cell population at day-2 and day-1 were similar to the postperitonitis control effluent. However, immunophagocytosis and phagocytosis capacity of the peritoneal macrophages was decreased in five of six episodes at day-2 and -1 compared to control peritoneal macrophages. The overnight effluents of day P revealed a moderate influx of neutrophilic granulocytes and an increase of bacterial killing capacity and chemotactic activity. Activation of the peritoneal T cells at day P was shown by the increase in MHC class II positive T cells and an increase in the CD4/CD8 ratio. Bacterial cell cultures of the effluents were positive in three episodes 24-48 h before peritonitis, and of all overnight effluents at day P. These results indicate that malfunctioning of phagocytosis by peritoneal macrophages may contribute to the development of a CAPD peritonitis.

Interleukin-8 production by human peritoneal mesothelial cells in response to tumor necrosis factor-alpha, interleukin-1, and medium conditioned by macrophages cocultured with Staphylococcus epidermidis.

Patients treated with continuous ambulatory peritoneal dialysis (CAPD) may suffer from recurrent peritonitis episodes caused by Staphylococcus epidermidis. Early recruitment of granulocytes from the peripheral blood is important for the peritoneal antibacterial defense of CAPD patients. In this study, human peritoneal mesothelial cells were shown to produce high levels of interleukin-8 (IL-8) in response to IL-1 beta and tumor necrosis factor-alpha (TNF alpha) but not lipopolysaccharide or S. epidermidis. Coculture of peritoneal macrophages with S. epidermidis induced high levels of IL-1 alpha, IL-1 beta, and TNF alpha in 24-h-conditioned medium. Preincubation of this medium with anti-TNF alpha, anti-IL-1 alpha, or anti-IL-1 beta partially blocked stimulation of IL-8 production by mesothelial cells. Added together, these antibodies abolished IL-8 production to a level just above background. Migration of granulocytes to the stimulated mesothelial cell-conditioned medium could be totally blocked with rabbit polyclonal anti-IL-8 antibody. Thus, mesothelial cells are important for the recruitment of granulocytes into the peritoneal cavity.

Antigen-presenting capacity of macrophages and dendritic cells in the peritoneal cavity of patients treated with peritoneal dialysis.

In this study the antigen-presenting capacity of human peritoneal cells and the influence of continuous ambulant peritoneal dialysis (CAPD) were studied. On average 6% of the peritoneal cells were dendritic cells (DC), with no difference between CAPD and control peritoneal cells. DC were enriched by selecting for non-adherent, Fc receptor-negative, low density cells. A typical spot-like CD68 positivity was seen in DC, in contrast to the pancytoplasmic staining pattern in macrophages. Peritoneal DC morphologically and functionally showed features of cells belonging to the DC lineage. Peritoneal DC were superior antigen-presenting cells for both allo-antigen, and Candida albicans antigen or purified protein derivative. CAPD peritoneal macrophages were two- to three-fold better stimulator cells for allogeneic T cells compared with control macrophages. The level of integrins/adhesins or MHC class I or II, as measured semi-quantitatively on the FACS, could not account for this phenomenon. In addition, a double chamber system showed that dialysate-activated macrophages produced soluble factors that could enhance DC-induced allogeneic T cell proliferation. In conclusion, human peritoneal cells contain a relatively high percentage of classical DC. CAPD treatment does not impair the antigen-presenting capacity of peritoneal cells, but instead upregulates the allo-antigen-presenting capacity of peritoneal macrophages.

Immuno-effector characteristics of peritoneal cells during CAPD treatment: a longitudinal study.

Peritoneal cells (PC) from 75 patients were immuno-phenotypically and functionally characterized during the first year of CAPD treatment (PCcapd) and compared to PC obtained by laparoscopy of healthy women (control peritoneal cells). Patients were divided, according to their peritonitis incidence (PI), into a high PI (HPI) and a low PI group (LPI). The yield of PCcapd decreased significantly over the year. The differential cell count and immunophenotype of PCcapd remained unchanged in the LPI group, but the percentage of macrophages decreased over the year in the HPI group. Macrophages in the PCcapd, when compared to control peritoneal cells, had a less mature phenotype as measured by RFD7 expression but a higher Fc-receptor expression. The PCcapd showed a higher percentage of B cells, CD4 positive T cells and activated T cells bearing HLA-DR/DQ when compared to the control peritoneal cells. Over the year a decrease in chemotactic activity of the PCcapd towards 10(-8) M N-formylmethionyl-leucyl-phenylalanine and dialysis effluent was observed in LPI patients but not in HPI patients. After one year of treatment, a significantly higher percentage of phagocytosing macrophages in the PCcapd of HPI patients was found when compared to LPI patients. During the year there was an increase of immunophagocytosis of PCcapd independent of PI. In conclusion, the CAPD peritoneal cellular immune system showed signs of both immaturity and activation. The decrease in the yield and in the chemotactic activity of PCcapd suggests an adaptation to the chronic stimulus of the dialysis fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adherence of Staphylococci to plastic, mesothelial cells and mesothelial extracellular matrix.

In this study we have investigated whether mesothelial cells (MC) and mesothelial extracellular matrix (ECM) are suitable substrates for the adherence of Staphylococci. Mesothelial cells were isolated from the peritoneal dialysis effluent by making use of their lack of Fc-receptors and capacity to attach firmly to plastic. After 10 days post-confluency the MC monolayer was removed with 0.1% Triton-X100 and the presence of an ECM shown by enzyme linked immunosorbent assay (ELISA). The ELISA showed the presence of fibronectin and laminin but not of type IV collagen and vitronectin. Bacterial adherence assays with Staphylococcus aureus (N:3 isolates) adhered well to both ECM (33.4%) and MC monolayers (40.2%; ECM vs. MC monolayers p < 0.03). Staphylococcus aureus adhered significantly better to both ECM (p < 0.05) and MC monolayers (p < 0.05) when compared to plastic. Staphylococcus epidermidis (N:3 isolates) showed similar adherence for plastic (22.1%) and MC monolayers (23.5%); mesothelial ECM was a relatively poor substrate for adherence (6.8%, p < 0.03). In conclusion, results obtained sofar do not indicate an increased risk for adherence of Staphylococci when the mesothelial ECM is exposed.

Monoclonal antibody EBM11 (anti-CD68) discriminates between dendritic cells and macrophages after short-term culture.

Identification of dendritic cells (DC) is usually done on the basis of their strong MHC class II expression, their typical dendritic morphology and their capacity to induce a strong proliferation of allogeneic T cells. However using these criteria DC can easily be confused with MHC class II positive macrophages (M phi). In addition, the lack of an antibody directed to a specific DC marker greatly hampers the discrimination between DC and M phi. In the present study it is shown that EBM11 (anti-CD68) is a marker specific for both human M phi and DC but in a distinctive way. Human DC locate the EBM11 reactivity in a discrete juxtanuclear spot in contrast to M phi which show EBM11 reactivity throughout the cytoplasm. This greatly improves identification of DC. Light and electron microscopy showed that the CD68 epitope is associated with (phago-)lysosomes. Remarkably the EBM11 spot was only seen in DC after short-term culture, which is an essential step in all classical DC enrichment procedures. Before culture, M phi and DC were indistinguishable. These results show the close relationship between M phi and DC and suggest an important role for the structure of the lysosomal apparatus in these antigen-presenting cells.

Chemotaxis of the peritoneal cells and the detection of a chemo-attractant in the effluent from peritoneal dialysis patients.

The migration of peritoneal cells from 25 continuous ambulatory peritoneal dialysis patients and eight healthy women undergoing laparoscopy were studied. Peritoneal cells of continuous ambulatory peritoneal dialysis patients migrated to commonly used chemoattractants, like N-formyl-methionyl-leucyl-phenyl-alanine-methyl- ester and casein, but they also migrated to high concentrations of recombinant interleukin-1 alpha and to endotoxin (lipopolysaccharide). In the peritoneal effluent from continuous ambulatory peritoneal dialysis patients a rather heat stable chemoattractant was found with a molecular weight of 40-200 kDa with an optimal activity at approximately 125 kDa. The chemoattractant is a protein and is not complement factor 5a or interleukin-1 and was only found in peritoneal effluent from continuous ambulatory peritoneal dialysis patients, but not in peritoneal fluid from healthy women undergoing laparoscopy. Therefore, peritoneal dialysis might induce the generation of a chemoattractant. The optimal chemotactic response of peritoneal cells from continuous ambulatory peritoneal dialysis patients to N-formyl-methionyl-leucyl-phenyl-alanine-methyl- ester in medium could be enhanced by replacing the medium by peritoneal effluent. So the chemotaxis of peritoneal cells to the factor in the peritoneal effluent is caused by another mechanism, which might involve different cell surface receptor populations, than the chemotactic response to N-formyl-methionyl-leucyl-phenyl-alanine-methyl-ester.

Recombinant gamma interferon induces class II major histocompatibility complex antigens on insulinoma cells.

Aberrant expression of major histocompatibility (MHC) antigens has been implicated as a factor contributing to organ-specific autoimmunity, such as progressive loss of pancreatic beta cells in type 1 diabetes. We investigated the potential of a rat beta cell tumour, RINM5F, to express enhanced levels of MHC antigens in vitro. To this purpose we treated RINM5F cells in vitro with recombinant rat gamma interferon (rIF gamma). We used monoclonal antibodies to RT1.A (class I) and RT1.B (class II) antigens of the rat MHC in conjunction with flowcytometry and immunoperoxidase techniques to analyse the expression of MHC antigens. Untreated RINM5F cells express low levels of RT1.A, whereas they are negative for RT1.B. Treatment with rIF gamma appeared to increase the expression of RT1.A antigens substantially. Most importantly, RT1.B antigens were newly expressed by rat insulinoma cells in vitro after treatment with rIF gamma. To our knowledge this is the first documentation of the potential of beta cells or their derivatives to express class II MHC antigens following IF gamma-treatment. This mechanism may play an important role in the augmentation and perpetuation of insulitis leading to type 1 diabetes mellitus.

Clinical time-course and characteristics of islet cell cytoplasmatic antibodies in childhood diabetes.

Circulating islet cell antibodies (ICA) were present in high frequency (80%) early after diagnosis and decreased in the time course of childhood diabetes mellitus. The complement fixing ability of islet cell antibodies (CF-ICA) in the course of the disease appeared to depend on the titre of ICA: the coefficient of correlation between ICA and CF-ICA titres was 0.79 and all ICA's with a titre over 16 were complement-fixing. Incubating fresh frozen human pancreatic sections thrice rather than once with the children's sera, increased the detectability of complement fixation by a factor 1.4 in all ICA-positive sera. Thus tested, the detection of complement fixation per se did not appear to have a separate pathogenic significance, as the fraction of complement fixing ICA's was almost constant throughout the clinical course. The presence of ICA-IgG subclasses also was dependent on the ICA titre: above a titre of 16 mostly all four subclasses could be detected. Incubating the pancreatic tissue thrice rather than once with ICA-positive sera resulted in enhanced detectability of ICA-IgG1. Early in the course of childhood diabetes, including two prediabetic children, most of the IgG subclasses could be detected in ICA, but after a duration of one year IgG1 alone was mainly seen. In two other children, having a family history of insulin-dependency, restriction to the IgG2 subclass was found.

Analysis of islet cell antibodies on frozen sections of human pancreas.

The sensitivity and specificity of the assay for islet cell cytoplasmic antibodies in human serum were examined using cryostat sections from fresh frozen pancreas. The specificity of the assay was close to 100% while the sensitivity was 40%-98% depending on the pancreas used. Inter-observer variation was 12-27%. End-point titres of islet cell antibodies varied with the sensitivity of each pancreas. End-point titration of the antibodies in two different laboratories using the same pancreas was significantly correlated (Spearman test p less than 0.001). We conclude that a reliable determination of islet cell antibody titres in human serum requires careful characterization of the sensitivity and specificity of each pancreas used as a source of frozen sections, in the indirect immunofluorescence assay.