Carin E. G. Havenith

Talc-induced inflammation in the pleural cavity

Talc administration into the pleural cavity induces pleurodesis. To obtain further insight into the inflammatory process that causes pleurodesis, the cellular kinetics in the pleural space after the administration of talc was studied, along with its relation to chemokine concentrations in the pleural fluid. Thirteen consecutive patients with idiopathic spontaneous pneumothorax and eight patients with malignant pleural effusions received talc pleurodesis. The first group was treated with talc poudrage, whereas the second group was treated with talc slurry. Pleural fluids were isolated before talc administration as well as 3, 6, 24, 48 and 72 h afterwards. The talc induced a rapid polymorphonuclear neutrophil (PMN) influx followed by an accumulation of macrophages. In addition, increased production of interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 was observed. The talc-induced PMN influx reached its maximum after 3-24 h, and was related to the IL-8 concentration. In contrast, the MCP-1 was not related to the macrophage accumulation. Talc-induced inflammation in patients with idiopathic spontaneous pneumothorax and malignant pleural effusion is characterized by an influx of polymorphonuclear neutrophils related to interleukin-8, followed by an accumulation of monocytes.

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.

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.

Antigen Specific T Cell Priming in Vivo by Intratracheal Injection of Antigen Presenting Cells

In the lung alveolar macrophages (AM) are the first cells to encounter pathogens and airborne antigens. In general, AM function poorly as accessory cells and even inhibit T cell proliferation in vitro 1,2. In previous experiments it was found that the increased numbers of dendritic cells (DC) in the bronchoalveolar lavages after intratracheal instillation of Bacillus Calmette-Guerin (BCG) were responsible for the antigen presentation in vitro 2. The AM suppressed the T cell proliferation in a concentration dependent manner2. Many cell types are capable of presenting antigen to T cells that already have undergone priming in situ, whereas the DC has been shown capable of delivering exogenous antigens directly to naive T cells in vitro 3.

Dendritic Cells Isolated From Rat and Human Non-Lymphoid Tissue are Very Potent Accessory Cells

In the past it was shown that dendritic cells (DC) are the in vitro equivalent of interdigitating cells present in T-cell area’s of lymphoid organs (1). Recently it has been shown that DC can also be isolated from rat normal steady state peritoneal cells (PC) and that these numbers can be increased after intraperitoneal injection of BCG (2, 3). The same can be demonstrated from rat broncho-alveolar lavages after installation of BCG (4). Finally DC can be isolated from the PC population in human, especially from CAPD (continuous ambulant peritoneal dialysis) patients (5), which represent a chronic inflammatory state (6).

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.

HETEROGENEITY OF DENDRITIC CELLS AND NOMENCLATURE

The Dendritic Cell System (DCS) comprises Dendritic Cells (DC) isolated from lymphoid organs and related in vivo equivalents such as interdigitating cells (IDC), which are present in thymus dependent areas of lymphoid tissues, and in the medulla of the thymus. However relatives of DC have also been isolated from other tissues and compartments such as epidermal Langerhans cells (LC), and all these cells share the functional capacity to present antigen to T cells.