Functional analysis of human bronchial mucosal T cells extracted with interleukin-2.

Functional analysis of T cells from the bronchial mucosa has been limited by difficulties in extracting T cells from this tissue. Because interleukin-2 (IL-2) is chemotactic for T cells, we determined whether this cytokine could be used to extract T cells from human bronchial wall (BW). Fresh tissue was obtained from 21 patients undergoing surgery for malignancy. Within the BW, 95% of T cells stained for the memory/activation marker CD45RO. When BW sections were incubated with IL-2 for 24 h, 88 to 91% of T cells emigrated into the culture medium. Compared with autologous blood T cells (also exposed to IL-2), these BW T cells expressed CD2 at a greater intensity and showed a fourfold reduction in cloning efficiency in response to phytohemagglutinin, and T-cell clones derived from the BW population displayed a tendency for higher interferon-gamma production. Furthermore, we were also able to extract and clone T cells from bronchoscopic biopsies in four subjects, suggesting that this method will provide a new avenue for examining T-cell function in airway inflammatory diseases.

Studies on the surface phenotype and functions of dendritic cells in parenchymal lung tissue of the rat.

Immunohistochemical analysis of frozen sections of rat lung tissue identified a widely distributed population of highly pleiomorphic Ia+ cells in alveolar septal walls, which are negative for the pan-macrophage marker ED4 and the related markers ED1, ED2 and ED9. Semi-purified dendritic cells (DC) prepared from lungs of rats exposed to an aerosol of ovalbumin (OA) triggered modest levels of proliferation of OA-immune T cells in vitro, demonstrating the potential of these cells in surveillance for inhaled antigens in vivo. Lung wall also exhibited modest stimulatory activity in mixed lymphocyte/leucocyte reaction (MLR) assays. Overnight incubation of the DC in T-cell culture supernatant markedly increased their T-cell stimulatory properties, concomitant with increased expression of Ia. These results suggest that analogous to epidermal Langerhans' cells, lung wall DC can effectively bind inhaled antigens in situ, but require additional maturation/activation signals before they can efficiently present the antigen to T cells.

Studies on the density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC) in the conducting airways.

Conventional immunohistochemical analysis of airway intraepithelial class II major histocompatibility complex (Ia) expression demonstrates a morphologically heterogeneous pattern of staining, suggestive of the presence of a mixed population of endogenous antigen presenting cells. Employing a novel tissue sectioning technique in conjunction with optimal surface antigen fixation, we now demonstrate that virtually all intraepithelial Ia staining throughout the respiratory tree in the normal rat, can be accounted for by a network of cells with classical dendritic cell (DC) morphology. The density of DC varies from 600-800 per mm2 epithelial surface in the large airways, to 75 per mm2 in the epithelium of the small airways of the peripheral lung. All the airway DC costain for CD4, with low-moderate expression of a variety of other leukocyte surface markers. Both chronic (eosinophilic) inflammation and acute (neutrophilic) inflammation, caused respectively by inhalation of chemical irritants in dust or aerosolised bacterial lipopolysaccharide (LPS), are shown to be accompanied by increased intraepithelial DC density in the large airways (in the order of 50%) and up to threefold increased expression of activation markers, including the beta chain of CD11/18. The kinetics of the changes in the DC network in response to LPS mirrored those of the transient neutrophil influx, suggesting that airway intraepithelial DC constitute a dynamic population which is rapidly upregulated in response to local inflammation. These findings have important theoretical implications for research on T cell activation in the context of allergic and infectious diseases in the respiratory tract.

Immunoregulation of asthma: control of T-lymphocyte activation in the respiratory tract.

Allergic asthma results ultimately from inappropriate responses to non-pathogenic airborn antigens by the respiratory tract T-cell system. In order to understand how sensitization to inhaled antigens develops, it is necessary to precisely define the tissue microenvironments within which T-cells occur in the respiratory tract, and to analyse the factor(s) which regulate their local activation. This review focuses upon recent data on the distribution of T-cells and antigen presenting cells within the lung and airway tissues of man and experimental animals, and examines the available information on their responses to antigenic stimulation. We argue that in the steady state, respiratory tract T-cells are tightly regulated by a series of inherent and acquired immunosuppressive control mechanisms, which normally limit local T-cell activation to situations where antigenic exposure is accompanied by an inflammatory stimulus, such as the case with incoming pathogenic microorganisms.

A contiguous network of dendritic antigen-presenting cells within the respiratory epithelium.

This study utilises a simple technique to section airway epithelium in a plane parallel to the basement membrane, thus providing a unique plan view of the intra-epithelial cell populations. Immunoperoxidase staining of these tissue sections for class II major histocompatibility complex Ia antigen reveals a virtually contiguous network of Ia-positive dendritic cells (DC) within the epithelium. These DC are shown to be capable of binding inhaled antigens in vivo in a form suitable for presentation to T cells. The strategic location of these cells and the fact that they account for virtually all staining in the airway epithelium during the steady state is convincing evidence that the DC network functions as the 'first line of defence' in surveillance for inhaled antigens and further suggests a major role for the intra-epithelial DC in allergic and infectious disease(s) in the respiratory tract.

Dendritic cells in the respiratory tract.

Studies from several laboratories on lung tissue samples from human and experimental animals have identified Ia+ cells with characteristic pleiomorphic (dendritic) morphology in the epithelium and underlying connective tissue, in both the conducting airways and in the distal lung. These dendritic cells (DC) are particularly prominent within the airway epithelium, forming a contiguous network equivalent to the Langerhans cells network of the epidermis. They may be readily concentrated from enzymatically disrupted respiratory tract tissue samples on the basis of their physical properties (notably non-adherence, lack of Fc-receptors and ultra-low density on percoll), and function as highly effective antigen presenting cells in vitro. Evidence is also accumulating that respiratory tract DC populations respond dynamically to local tissue inflammation, and as such may play a prominent role in immunoinflammatory disease processes in the airways and the distal lung.

Ia-positive dendritic cells form a tightly meshed network within the human airway epithelium.

In this report we have employed an alternative tissue-sectioning procedure which provides a plan view of intra-epithelial cell populations within the airway wall. Immunoperoxidase staining of such sections for class II MHC (Ia) antigen revealed the presence of a highly developed intra-epithelial network of Ia-positive dendritic cells, which was not evident employing conventional cross- or longitudinal tissue sections. This finding has important implications for the study of mechanisms underlying allergic and infectious diseases of the respiratory tract.

MHC class II antigen-bearing dendritic cells in pulmonary tissues of the rat. Regulation of antigen presentation activity by endogenous macrophage populations.

Collagenase digestion of tissue slices from perfused, lavaged SPF rat lung released approximately 10(8) viable mononuclear cells per gram tissue, which comprised 35% T lymphocytes and up to 26% macrophages. A subset of these cells that were Ia+, surface Ig-, nonadherent, FcR- and of ultra low density (putative dendritic cells [DC]), presented protein antigen to immune T cells in vitro, and this function was inhibited by the presence of low numbers of endogenous adherent, FcR+ cells (putative macrophages). APCs were also identified in digests from tracheal epithelium, and were shown to bind antigen in immunogenic form as a result of natural (inhalation) exposure in vivo. Immunoperoxidase staining of frozen sections revealed populations of strongly Ia+ cells with prominent DC-like morphology within the alveolar septal walls and the tracheal epithelium; in both areas, they were closely associated with pleiomorphic cells that expressed macrophage surface markers. We accordingly postulate that interactions between Ia+ antigen-presenting DCs and endogenous tissue macrophages play an important role in regulating T cell activity in the respiratory tract.

Localization of T cells, macrophages and dendritic cells in rat respiratory tract tissue: implications for immune function studies.

Monoclonal antibodies against a range of surface markers were used to localize T cells, macrophages and dendritic cells (DC) in sections of rat trachea and peripheral lung, employing the immunoperoxidase technique. A population of Ia-bearing cells with characteristic DC morphology was identified within the tracheal epithelium, closely associated with the basement membrane, and Ia+ dendritic processes from these cells penetrated the epithelium reaching the overlying fluid layer. A second DC-like population, also Ia+ but differing from the airway DC in expression of other markers, was identified within the alveolar septal walls. Both types of DC were intimately associated with populations of pleiomorphic heterogenously staining macrophages. In addition, a large population of T lymphocytes was identified within the alveolar septa; the lymphocytes occurred as single, isolated cells, distributed randomly throughout the lung parenchyma.

Ia antigens in rat kidney, with special reference to their expression in tubular epithelium.

The distribution of Ia antigens in rat kidneys has been investigated by an immunoperoxidase technique, using monoclonal antibodies directed at framework determinants on molecules equivalent to gene products of the I-A and I-E/C regions of the mouse major histocompatibility complex (MHC). Antigens mapping to both loci were detected in proximal convoluted tubule epithelium and on scattered dendritic cells in the interstitial connective tissues, glomeruli, and mucosa of the renal pelvis. All other structures were negative. Genetic studies indicate that the levels of expression of Ia molecules from both genetic loci are controlled by non-MHC genes in epithelium, but not on lymphocytes. The subcellular distribution of Ia molecules has been investigated in tubular epithelium and is discussed in relation to their possible functions in epithelia.

Antigen presentation by macrophages from adult thymectomised mice.

Recent claims that macrophages from adult thymectomised mice fail to mature to a stage where they can 'present' antigen were examined. Comparisons were made between the capacity of antigen-pulsed peritoneal exudate macrophages from adult thymectomised and normal mice to elicit both primary and secondary lymphoproliferative responses in vitro, and to prime lymph node cells for antigen-specific recruitment in vivo. Macrophages from thymectomised donors were partially deficient in their capacity to activate naive or immune T-cells in vitro but were nevertheless capable of emitting a significant immunogenic signal. These results suggest partial, but not absolute T-cell regulation of the immunogenic function(s) of exudate macrophage populations.

Improved method for the isolation of purified mouse peritoneal macrophages.

Mouse peritoneal macrophages were cultured for 45 min in medium supplemented with fetal calf serum (FCS) is petri dishes coated overnight with heat-inactivated FCS. After removal of non-adherent cells by washing, adherent cells were detached by a brief incubation in the presence of sub-toxic levels of ethylenediamine tetraacetate (EDTA). Overall peritoneal macrophage recoveries of 90% can be routinely achieved with this method, and full cell viability is maintained.