Separation of alveolar macrophages and dendritic cells via autofluorescence: phenotypical and functional characterization.

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses. Flow cytometric parameters, density fractionation, and a series of ED monoclonal antibodies raised against rat macrophage antigens showed that both AMs and MDC were diverse populations. After overnight culture, more than 80% of an MDC population with a density range of 1.065-1.079 changed to a lower density (< 1.056) and morphologically developed into DCs with many processes. Concomitantly, monoclonal antibody ED1 expression changed from a granular pattern to a discrete juxtanuclear spot localization.

Rat monocyte-derived dendritic cells function and migrate in the same way as isolated tissue dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells and are therefore useful to induce immune responses against tumor cells in patients. DC can be generated in vitro from monocytes using GM-CSF and IL-4, the so-called monocyte-derived DC (MoDC). To achieve antitumor responses, MoDC must be able to migrate to the draining lymph nodes after injection to induce cytotoxic T cells. Therefore, we studied migration of MoDC in a rat model. Functional rat MoDC were generated from PVG-RT7B rats and injected subcutaneously into PVG rats. These rat strains differ only at one epitope of the leukocyte-common antigen, which can be recognized by the antibody His 41. The advantage is that migrated cells can be detected in the draining lymph nodes by staining sections with His 41+; thus, migration is not influenced by labeling procedures. Rat MoDC migrated to the T-cell areas of the draining lymph nodes, just as isolated Langerhans cells or spleen DC do. In contrast, monocytes also migrated to the B-cell areas and the medulla.

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.

Methods for studying immuno-effector functions and antigen presenting activity of human macrophages.

Macrophages are now recognized as cells that perform a variety of functions making them important cells in both the specific and non-specific immune responses. In the inflammatory response mature monocytes leave the bloodstream to enter macrophages and then migrate to the site of infection. This process is usually co-ordinated by the production of so-called chemotaxins that direct the movements of the macrophages. This can be measured in vitro by a chemotaxis assay that separates macrophages from the chemotactic fluid by a micropore filter. After incubation migrated cells are counted at the other side of the filter and indicate the chemotactic response. At the inflammatory site phagocytosis and killing of invading bacterial micro-organisms is an important feature of macrophages. We describe an assay to measure phagocytosing capacity using IgG-coated sheep red blood cells and a bacterial killing assay. In addition a tumor killing assay is briefly introduced. Finally, the specific immune response is initiated by presentation of antigen to antigen specific T cells by specialized cells such as dendritic cells that are closely related to macrophages. An enrichment procedure for macrophages and dendritic cells is outlined and antigen presentation assays are described.

Mouse microglial cell lines differing in constitutive and interferon-gamma-inducible antigen-presenting activities for naive and memory CD4+ and CD8+ T cells.

We developed a panel of non-virus transformed cell lines derived from individual microglial precursors residing in the brains of normal mice. These colony stimulating factor-1-dependent cell lines are B7-1+ (CD80), Mac-1+, Mac-2+, Mac-3+, CD45+, MHC class I+, colony stimulating factor-1 receptor+, and they ingest antibody-coated particles. However, the cell lines differ in their expression of B7-2 (CD86), F4/80, Ly-6C and MHC class II molecules. They also differ in their ability to constitutively process and present antigens to naive CD4+ and CD8+ T cells, memory CD4+ and CD8+, and in the manner by which interferon gamma modulates their antigen-presenting activities. These cell lines should be valuable as models for studies on the immunobiology of the microglia.

Effect of Bacillus Calmette-Guérin inoculation on numbers of dendritic cells in bronchoalveolar lavages of rats.

Dendritic cells (DC) are present in all lymphoid tissues and are widely distributed in the airway epithelium and lung parenchyma. In this study DC were morphologically and cytochemically identified in normal rat bronchoalveolar lavage (BAL), although in very low percentages. Furthermore, the total population as well as different Percoll density fractions demonstrated poor antigen-presenting capacity and even suppressed antigen-specific stimulation by rat splenic DC. In contrast, when an inflammatory response was induced by intratracheal inoculation of Bacillus Calmette-Guérin (BCG), an increase of Ia-positive cells, containing high percentages of monocytes and DC (MDC) was found. In BAL, DC increased about 25 times within 48 h after BCG inoculation. These BCG-induced BAL cells as well as the different density fractions showed a high antigen-presenting capacity at low concentrations. However, at higher concentrations they were suppressive, except for the highest density fraction which lacked alveolar macrophages (AM). These results indicate that the increased numbers of Ia-positive MDC during an inflammatory reaction are very likely responsible for antigen presentation in vitro. In contrast, AM suppress the antigen-specific T cell proliferation in a concentration dependent manner.

Live bacterial delivery systems for development of mucosal vaccines.

By expression of foreign antigens in attenuated strains derived from bacterial pathogens and in non-pathogenic commensal bacteria, recombinant vaccines are being developed that aim to stimulate mucosal immunity. Recent advances in the pathogenesis and molecular biology of these bacteria have allowed rational development of new and improved bacterial carriers and more effective gene expression systems. These advances have improved the performance and versatility of these delivery systems to induce mucosal immunity to recombinant antigens in animal models. Application of these (improved) technologies for development of human vaccines is still limited and awaits further exploration.

Isolation of human lung dendritic cells.

In the lung several cell types are capable of presenting antigen to T cells. The dendritic cells (DC) are the most potent antigen-presenting cell. DC form a rare cell population in the lung and early studies were hampered because scarce cell populations are seldom easy to isolate. Besides, this cell is phenotypically heterogeneous depending on its localization, differentiation, or activation status. This chapter will elaborate on the heterogeneity that has to be taken into consideration when studying and isolating lung DC. Furthermore, techniques for the isolation of lung DC from human parenchymal lung tissue and bronchoalveolar lavage fluid (BAL) are described.

Effect of peritoneal dialysis fluid measured in vivo in a rat-model of continuous peritoneal dialysis.

To study the long-term effects of dialysis fluids on the peritoneal cavity, an in vivo model for continuous peritoneal dialysis in rats was developed. Mini vascular access ports were implanted subcutaneously in the neck of the rats and an attached catheter was instilled into the peritoneal cavity. Rats were injected daily with 10 mL of standard 3.86% Dianeal or saline for a period up to 12 weeks. In the peritoneal cavity an initial increase in total cells was observed after 4 weeks of fluid instillation. This had declined after 12 weeks. A similar trend was also seen for macrophage and neutrophil numbers, whereas the percentage of lymphocytes kept increasing in time. An effect of fluid instillation was observed on the density and the morphology of the mesothelial monolayer of the rats. A higher density of cells was observed after 12 weeks, and foci of young mesothelial cells within activated mesothelium were found. The results show that the rat model presented can be compared with the situation in the peritoneal cavity of continuous ambulatory peritoneal dialysis (CAPD) patients, and therefore is suitable for intervention studies.

Mouse resident microglia: isolation and characterization of immunoregulatory properties with naïve CD4+ and CD8+ T-cells.

We describe a non-enzymatic procedure designed to isolate in high purity resident microglia from the brains of normal mice. This procedure allowed for the characterization of the cells without concern that their surface features had been enzymatically altered during tissue processing. A cell population was obtained and judged to consist primarily of microglia because essentially all the cells were Mac-1+, Mac-3+, F4/80+, CD44+, CD54+, and CD86+, and they expressed CD45 with a mean fluorescence intensity value of about one-half that of tissue macrophages. The cells also expressed marginal levels of MHC class I, CD14, CD40, and CD80, but lacked detectable MHC class II, CD4, CD8, CD45R, and CD102 molecules. Molecular phenotyping revealed that the purified microglial population contained mRNA transcripts encoding the receptor for colony stimulating factor-1 (CSF-1), the macrophage growth factor, and contained few, if any, transcripts for glial fibrillary acidic protein, an astrocyte-specific marker. Ex vivo, the microglia constitutively stimulated, in the mixed leukocyte reaction, the proliferation of naive allogeneic CD8+, but not CD4+ T-cells. However, they failed to present a protein antigen to naive antigen-specific CD4+ T-cells unless pretreated with interferon-gamma, a response that was inhibited by antibodies to CD86. Agar-cloning experiments confirmed that normal mouse brain contains a CSF-1-responsive cell that gave rise to cells with identical immunophenotypic characteristics as freshly isolated resident microglia. Moreover, the microglial progenitor cell located in a density fraction that was different from that containing the mature resident microglia.

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.

Recombinant Lactobacillus plantarum inhibits house dust mite-specific T-cell responses.

Recent evidence suggests that chronic exposure to lactobacilli, which are part of the normal intestinal flora, inhibits the development of allergic disorders. Allergy is mediated by Th2 cells, which produce high levels of IL4 and IL5, and suppressive effects of lactic acid bacteria on the development of allergy have been attributed to their Th1-inducing properties. On the other hand, lactic acid bacteria have also been shown to suppress autoimmune disorders which are mediated by Th1 cells producing high levels of IFNgamma. To study this apparent discrepancy, the immunomodulatory potential of lactobacilli was evaluated using recombinants that express an immunodominant T-cell epitope of Der p 1 of house dust mites. Mucosal immunization of C57BL/6 J mice with such recombinants resulted in the induction of T cells which produced low amounts of IFNgamma. Immunization with the house dust mite peptide followed by treatment with recombinant Lactobacillus plantarum resulted in the inhibition of both IFNgamma and IL5 production. The effect on IFNgamma production was shown to be a non-specific effect of L. plantarum. The effect on IL5 production, however, was only observed when the recombinant expressing the Der p 1 peptide, but not the control recombinant, was used for treatment. Neither of the recombinants had an effect on the antibody response. Taken together, these data suggest that recombinant L. plantarum may be a suitable candidate for the treatment of allergic disorders.

Migration of dendritic cells into the draining lymph nodes of the lung after intratracheal instillation.

The migration of dendritic cell (DC)-enriched populations and alveolar macrophage (AM) populations isolated from PVG RT7.2 rats was studied after local administration to recipient PVG RT7.1 rats. The monoclonal antibody His41, which is directed against the common leukocyte antigen of the RT7.2 rat, was used to detect migrated cells. Injection of the splenic DC and AM subcutaneously into the footpads resulted in migration of both cell types to the popliteal lymph nodes after 24 h. DC located predominantly in the T cell-dependent areas, whereas AM located more in the medulla and medullary cords and spread throughout the outer cortex area. After intratracheal instillation of splenic DC, these cells were found predominantly in T cell-dependent areas of the draining lymph nodes of the lung after 24 h. In contrast, AM did not migrate to the draining lymph nodes after intratracheal instillation. Combined with those from earlier studies, these data show that DC present in the alveolar lumen may pick up airborne antigen and migrate to the draining lymph nodes of the lung, where they can induce primary T cell responses.

T cell priming in situ by intratracheally instilled antigen-pulsed dendritic cells.

In the present study, splenic dendritic cells (DC) and alveolar macrophages (AM) were pulsed with antigen in vitro and subsequently intratracheally instilled to test whether these cells have the capacity to sensitize T cells in the draining lymph nodes of the lung. The data demonstrate that antigen-pulsed DC, instilled in the bronchoalveolar lumen, induce antigen-specific T cell priming in vivo in the draining lymph nodes. T cell priming is only seen with viable but not with killed antigen-pulsed DC. Amounts as low as 5 x 10(3) to 10 x 10(3) cells can still induce some responsiveness. In addition, it was found that instillation of viable as well as killed pulsed Ia-negative AM also leads to T cell priming, although about 10 times higher numbers of cells had to be used in comparison with DC. The results suggest that DC instilled in the bronchoalveolar lumen present antigen directly to naive T cells, whereas for AM other mechanisms are involved.

Laparoscopic surgery preserves monocyte-mediated tumor cell killing in contrast to the conventional approach.

BACKGROUND: Experimental animal research shows that immunologic defenses against tumor cells are disturbed by surgical trauma, resulting in an increased rate of tumor implantation and the growth of subsequent metastases. Minimally invasive surgery is associated with a preservation of postoperative immunologic functions and, in animal models, with decreased tumor growth. The objective was to study the influence of several surgical procedures, approached conventionally and laparoscopically, on interleukin-6 (IL-6) and monocyte-mediated cytotoxicity (MMC). METHODS: Five groups of five patients each were included in this prospective study: laparoscopic cholecystectomy (minor trauma) group, Nissen fundoplication (laparoscopic and conventional as moderate trauma) groups, and sigmoid colectomy (laparoscopic and conventional as major trauma) groups. Preoperatively, 1 and 4 days after surgery, IL-6 and MMC against SW948 colon cancer cell line were determined. RESULTS: The IL-6 levels differed significantly between the three laparoscopic procedures (p = 0.004) and increased according to the degree of trauma. There was no significant difference in MMC between the three laparoscopic procedures. However, MMC was suppressed after conventional procedures and preserved after laparoscopic procedures (p = 0.001). There was no correlation between IL-6 levels and changes in MMC. CONCLUSIONS: More extensive laparoscopic procedures induce increased levels of IL-6, reflecting higher levels of trauma. Conventional surgical procedures result in depressed MMC in the postoperative period. After laparoscopic procedures, MMC is preserved. These findings may be of importance in preventing implantation and growth of cancer cells spread by surgical manipulation.

Dendritic cells and their precursors isolated from human bronchoalveolar lavage: immunocytologic and functional properties.

Human bronchoalveolar lavage (BAL) has been described to contain, besides a large number of alveolar macrophages (AM) (approximately 95%), small numbers of monocyte-like cells (approximately 2%) and dendritic cells (DC) (approximately 0.4%). To separate AM (high autofluorescence) from DC, we used a fluorescence activated cell sorter (FACS) to separate BAL cells into a low autofluorescent (LAF) fraction and a high autofluorescent (HAF) fraction. Immunocytologic and functional properties of these fractions were investigated. The LAF fraction was composed of acid phosphatase (APh)- and RFD9-negative cells, which were strongly positive for HLA-DR, L25, RFD1, and CD68. A portion of these cells expressed CD1a (22%) and My4 (60%). The marker pattern of these cells is reminiscent to that of intraepithelial bronchial DC and to that of blood DC. The majority of the LAF cells had a monocyte-like morphology, but after overnight culture the percentage of LAF cells with long cytoplasmic extensions (DC morphology) was strongly augmented (from 18 to 51%). The HAF fraction contained 100% AM, strongly positive for APh, HLA-DR, CD68, RFD7, and RFD9. In culture, the LAF cells formed clusters with T cells and vigorously stimulated the proliferation of allogeneic T cells and naive (CD45RO-negative) T cells. BAL and LAF cells produced higher responses in nonsmokers than in smokers. In contrast, HAF cells did not form clusters with T cells and did not stimulate allogeneic T cell proliferation. HAF cells even suppressed mitogen-driven T cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)